Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1997-05-22
2001-02-20
Venkat, Jyothsna (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S007100, C435S007210, C435S007230, C435S320100, C530S300000, C530S344000, C530S399000, C530S412000, C530S413000
Reexamination Certificate
active
06190856
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides improved assays for detection of proteins utilizing virus expressing a ligand for the protein. Such assays can be used in areas wherein traditional immunoassays have previously been utilized.
2. Background Art
Conventional immunoassays rely on the high affinity specific interaction between immunoglobulin and its corresponding epitope. Generally, a variable region of immunoglobulin forms a binding pocket that recognizes either conformation or primary sequence of a given epitope. Because the epitope can be a very small and linear region of a protein, it is common to find that an immunoglobulin recognizes a protein in either native or denatured conformation by binding to a small embedded peptide. Therefore detection agents that can recognize native conformations of peptides, such as receptors, channels, other surface proteins, and extracellular proteins, would be highly useful.
Use of random peptide phage libraries to isolate lead compounds for target proteins has yielded a variety of peptides that recognize specific target proteins with interesting biological activities. Such strategy has provided interesting binding peptides which recognize important proteins including antibodies (Smith, 1985; Cwirla et al., 1990; Scott and Smith, 1990; Kay et al., 1993), streptavidin (Devlin et al., 1990), calmodulin (Dedman et al., 1993), SH3 domain (Sparks et al., 1994), and endoplasmic reticulum protein BiP (Blond-Elguindi et al., 1993). Some of these peptides have yielded important insights into the structure-function of the target protein. For example, analysis of peptides that bind to BiP have shown that BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule (Blond-Elguindi et al., 1993).
N-methyl D-aspartate receptor (NMDAR) channels, found in brain, belong to the superfamily of ligand-gated ion channels, which become selectively permeable to ions upon binding to ligands. The glutamate-mediated channel activity is important for synaptic plasticity, synaptogenesis, and excitotoxicity. NMDA receptors are encoded by at least five genes (NR1, 2A, 2B, 2C, and 2D) and their spliced variants
7
. NR1 is an essential subunit for mediating glutamate-induced channel activity, since a homomultimeric NR1 complex, when expressed in Xenopus oocytes, exhibits some of key properties found in native channels. These properties include direct permeability of calcium, voltage dependent Mg
2+
blockade of the ion channel, and binding sites for modulators such as Zn
3+
, glycine, and polyamines. The other subunits by themselves cannot form a functional channel. However, they coassemble with NR1 to form heteromultimers which increase the expression level and channel diversity. The NR1 polypeptide can be divided into two regions: an extracellular amino-terminal domain containing the putative agonist binding site, and a hydrophobic core region with multiple transmembrane segments which participate the formation of the ion conducting pathway. Although several classes of compounds have been found to modulate the NMDA receptor, at the molecular level little is known in terms of amino acid residues that are responsible for the interaction. To better understand the physiological roles of NMDA receptors with different subunit composition or to detect NMDA receptors in samples, it would be helpfuil to develop specific ligands that are capable of distinguishing different subunits.
Excitotoxic mechanisms have a well established role in the pathogenesis of neuronal injury following acute CNS insults such as ischaemia and trauma Increasing evidence now supports their roles in chronic neurodegenerative disorders such as motor neuron diseases (MND). Although the molecular mechanisms underlying excitotoxic neuronal injury are still being elucidated, a large body of evidence indicates that the cascade of events resulting from elevation of intracellular free calcium is likely to play a major role, which is thought to be mediated by NMDA receptor and voltage-gated calcium channels. NMDA receptor has several very important properties of relevance to excitotoxicity, particularly, the ability to permeate calcium ions. Thus, understanding of NMDA receptor properties at the molecular level and development of reagents that specifically modulate these receptors can be of importance for understanding excitotoxicity resulted diseases and providing potential leads which could facilitate future treatments. By further improving the affinity and stability of NMDA receptor-specific ligands, their potential neuronal protective activity can be improved.
The present invention provides new procedures which use bacterial virus (including bacteriophage) as a detective reagent, in a way comparable to primary antibody in immnunoassays, to monitor the expression of receptor and channels in mammalian cells. Conversely, the phage binding described herein requires the corresponding receptor in proper conformation to present the binding pocket for the peptide to bind. Because the conformation of receptor binding pocket is critical for the peptide binding, peptide-receptor target interaction per se can have higher specificity than that of some antibody-receptor interaction. In search of novel peptides that modulate receptor activity, phage clones with specific interacting peptides from random peptide libraries have been isolated by panning selection, which is often based on the multivalent interaction between the phage particle and target receptor. These assays provide an improved way to detect protein expression and to study peptide-receptor interaction at the molecular level.
REFERENCES:
patent: 4851331 (1989-07-01), Vary et al.
patent: 5270170 (1993-12-01), Schatz et al.
patent: 5432018 (1995-07-01), Dower et al.
patent: WO 94/26787 (1994-11-01), None
Banner & Witcoff , Ltd.
Ponnaluri P.
The Johns Hopkins University
Venkat Jyothsna
LandOfFree
Methods of detection utilizing modified bacteriophage does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods of detection utilizing modified bacteriophage, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods of detection utilizing modified bacteriophage will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2605632