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Coppock M.B.,U.S. Army | Farrow B.,California Institute of Technology | Warner C.,Edgewood Chemical Biological Center | Finch A.S.,U.S. Army | And 4 more authors.
Proceedings of SPIE - The International Society for Optical Engineering | Year: 2014

Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design. © 2014 SPIE. Source

Pfeilsticker J.A.,California Institute of Technology | Umeda A.,California Institute of Technology | Farrow B.,California Institute of Technology | Hsueh C.L.,California Institute of Technology | And 4 more authors.
PLoS ONE | Year: 2013

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60oC. © 2013 Pfeilsticker et al. Source

Deyle K.M.,California Institute of Technology | Farrow B.,California Institute of Technology | Qiao Hee Y.,California Institute of Technology | Work J.,California Institute of Technology | And 7 more authors.
Nature Chemistry | Year: 2015

Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate. © 2015 Macmillan Publishers Limited. All rights reserved. Source

Farrow B.,California Institute of Technology | Hong S.A.,Gwangju Institute of Science and Technology | Romero E.C.,California Institute of Technology | Lai B.,Indi Molecular | And 7 more authors.
ACS Nano | Year: 2013

We report on a robust and sensitive approach for detecting protective antigen (PA) exotoxin from Bacillus anthracis in complex media. A peptide-based capture agent against PA was developed by improving a bacteria display-developed peptide into a highly selective biligand through in situ click screening against a large, chemically synthesized peptide library. This biligand was coupled with an electrochemical enzyme-linked immunosorbent assay utilizing nanostructured gold electrodes. The resultant assay yielded a limit of detection of PA of 170 pg/mL (2.1 pM) in buffer, with minimal sensitivity reduction in 1% serum. The powdered capture agent could be stably stored for several days at 65 C, and the full electrochemical biosensor showed no loss of performance after extended storage at 40 C. The engineered stability and specificity of this assay should be extendable to other cases in which biomolecular detection in demanding environments is required. © 2013 American Chemical Society. Source

Das S.,California Institute of Technology | Nag A.,California Institute of Technology | Liang J.,California Institute of Technology | Bunck D.N.,California Institute of Technology | And 19 more authors.
Angewandte Chemie - International Edition | Year: 2015

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies. © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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