Antibodies in Proteomics

Bradbury, Andrew; Velappan, Nileena; Verzillo, Vittorio; Ovečka, Milan; Marzari, Roberto; Sblattero, Daniele; Chasteen, Leslie; Siegel, Robert W.; Pavlik, Peter

doi:10.1385/1-59259-666-5:519

Cited by 4 publications

(4 citation statements)

References 114 publications

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“…The extracted total RNA was used as template for 5 0 -RACE-ready cDNA synthesis (Takara Bio, Mountain View, CA). The heavy chain variable domain (VH) and the light chain variable domain (VK) of 4D9 were amplified separately by PCR using the 5 0 -RACE adaptor-specific forward primer, rat IgG, or kappa constantspecific reverse primers (Bradbury, 2010). For VK domain amplification, a peptide nucleic acid oligo (CCTGTGGAGGAGGAG-GATGCT-KK) was used to selectively amplify the functional kappa chain (Cochet et al, 1999).…”

Section: Hybridoma Antibody Sequencingmentioning

confidence: 99%

Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

et al. 2020

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Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease‐associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full‐length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α‐secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α‐secretase‐mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho‐SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid β‐peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9‐bound. Moreover, in a mouse model for Alzheimer's disease‐related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease‐associated state.

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Section: Hybridoma Antibody Sequencingmentioning

confidence: 99%

Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

et al. 2020

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“…In the rapidly growing fields of proteomics and glycomics, in-depth analysis of protein expression, modification, and interaction on a genomic scale requires the development of specific and high-affinity reagents. − Traditionally, proteinogenic polymers, especially antibodies, have served in this role; however, they suffer from a number of shortcomings, ranging from poor stability and variable production quality to limited target availability and issues with specificity profiles. − Despite over 500 000 commercially available antibodies, researchers continue to express concerns over accessibility to high-quality antibodies for biomedical research. − These issues have stimulated the development of alternative technologies to generate high-affinity reagents for proteins that rival the performance of traditional antibodies. − …”

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confidence: 99%

A High-Fidelity Codon Set for the T4 DNA Ligase-Catalyzed Polymerization of Modified Oligonucleotides

Kong

Hili

2015

ACS Comb. Sci.

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In vitro selection of nucleic acid polymers can readily deliver highly specific receptors and catalysts for a variety of applications; however, it is suspected that the functional group deficit of nucleic acids has limited their potential with respect to proteinogenic polymers. This has stimulated research toward expanding their chemical diversity to bridge the functional gap between nucleic acids and proteins to develop a superior biopolymer. In this study, we investigate the effect of codon library size and composition on the sequence specificity of T4 DNA ligase in the DNA-templated polymerization of both unmodified and modified oligonucleotides. Using high-throughput DNA sequencing of duplex pairs, we have uncovered a 256-membered codon set that yields sequence-defined modified ssDNA polymers in high yield and with high fidelity.

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“…1). In the future, however, the alternative approaches of generating recombinant antibody molecules may well supplement or replace these strategies once brought to a comparable maturation (Bradbury, 2003;Bradbury et al, 2004).…”

Section: In Vitro Antibody Generationmentioning

confidence: 99%

Perspectives for systematic in vitro antibody generation

Konthur

Hust

Dübel

2005

Gene

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After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

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Antibodies in Proteomics

Cited by 4 publications

References 114 publications

Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

A High-Fidelity Codon Set for the T4 DNA Ligase-Catalyzed Polymerization of Modified Oligonucleotides

Perspectives for systematic in vitro antibody generation

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