Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-06-09
2001-08-07
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200
Reexamination Certificate
active
06270981
ABSTRACT:
BACKGROUND OF THE INVENTION
Leptin
Leptin, the product of the obese gene (1), is secreted by adipocytes, and functions as a peripheral signal to the brain that regulates food intake and energy metabolism. Leptin is thought to exert its action in the hypothalamus through its receptor, OB-R “the sequence of which is set forth in SEQ ID NO:1” (2). Rodents with mutations that prevent normal expression of either leptin or full-length OB-R are profoundly obese, diabetic, and have a reduced metabolic rate (3). However, human obesity does not appear to be associated with mutations in the genes encoding leptin or OB-R (4, 5). Although mice with a mutant obese gene can be returned to normal weight by administration of recombinant leptin (6-8), it seems unlikely that this approach will succeed in obese humans because their serum leptin levels are chronically elevated (9-11). Obese humans, therefore, appear to be “leptin resistant” (9, 12, 13) in that they do not generate a signal commensurate with their serum leptin levels, perhaps because of defective transport of leptin across the blood-brain barrier (14) or an inadequate OB-R response. Analysis of OB-R signaling pathways may reveal alternative therapeutic approaches of boosting OB-R responses to overcome leptin resistance and reverse obesity.
Leptin and OB-R are members of the four-helical bundle cytokine and receptor superfamilies respectively (2, 15). OB-R is most closely related to the gp130 signal transducing receptor that is activated by cytokines such as IL-6 (CNTF: ciliary neurotrophic factor; IL-6: Interleukin-6; Jak: Janus kinase; NT-3: neurotrophin-3; SH2: Src homology domain 2; STAT: Signal Transducer and Activator of Transcription; TOBR: Trk-C-OB-R chimeric receptor) and CNTF, whose signaling pathways have been intensively studied (16, 17).
Ligand binding induces either homodimerization of gp130, or heterodimerization of gp130 with related signal transducing receptors, leading to activation of the receptor-associated Jaks (18, 19). The Jaks then phosphorylate gp130 on cytoplasmic tyrosine residues, forming phosphotyrosine motifs that recruit specific SH2-containing signaling molecules such as STAT3, and the protein tyrosine phosphatase SHP-2 (previously known as PTP1D, SH-PTP2 or Syp) (20). Removal or mutation of the phosphotyrosine motifs in the receptor eliminates activation of the corresponding SH2 target molecule (20). Cytoplasmic deletions appear to affect OB-R in a similar manner: there are multiple isoforms of OB-R corresponding to alternatively spliced products with different cytoplasmic domains (2, 21-24), but only one isoform with several potential phosphotyrosine motifs, known as the long form or OB-Rb, appears capable of mediating leptin's weight controlling effects (21, 22, 25, 26). Obese diabetic mice have a mutation in OB-R that prevents expression of the long OB-R splice isoform, which renders them incapable of appropriately mediating leptin's actions (21, 22). The finding that only the long form of OB-R contains the sequence YXXQ (2), which is a motif that specifies STAT3 activation (20), raised the possibility that STAT3 is critical for mediating leptin responses. Recent results verify that STAT3 is activated both in cultured cells (25-27) and in vivo (28) by the long form of OB-R, and not by a truncated OB-R or the long form of OB-R with a mutant YXXQ motif (26, 29). Although leptin-induced activation of overexpressed STAT1 and STAT5b is also observed in transfected cells (25, 26), only activation of STAT3 has been detected in vivo upon stimulation of hypothalamic OB-R by administration of leptin (28). Thus it appears likely, but unproven, that transcriptional activation of target genes by STAT3 in the hypothalamus is a critical pathway that mediates leptin's regulation of food intake and energy metabolism.
Assay Systems
Ligand-receptor assays rely on the binding of ligands to receptors to determine the presence and/or concentration of ligands in a sample. Ligand-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize receptors in substantial excess over the concentration of ligand to be determined in the assay. Sandwich assays, in which the ligand is detected by binding to two receptors, one receptor labeled to permit detection and a second receptor frequently bound to a solid phase to facilitate separation from unbound reagents, such as unbound labeled first receptor, are examples of non-competitive assays.
Competitive assays generally utilize ligand from the sample, a ligand analogue labeled to permit detection, and the competition of these species for a limited number of binding sites provided by the ligand receptor. Those skilled in the art will appreciate that many variations of this basic competitive situation have been previously described. Examples of ligands which are commonly measured by competitive ligand-receptor assays include haptens, hormones and proteins. Antibodies or receptorbodies that can bind these classes of ligands are frequently used in these assays as ligand receptors.
Competitive ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays, all of the reactants participating in the competition are mixed together and the quantity of ligand is determined by its effect on the extent of binding between ligand receptor and labeled ligand analogue. The signal observed is modulated by the extent of this binding and can be related to the amount of ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive immunoassay in which the labeled ligand analogue is a ligand-enzyme conjugate and the ligand receptor is an antibody capable of binding to either the ligand or the ligand analogue. The binding of the antibody to the ligand-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound ligand and ligand-enzyme conjugate for antibody binding sites, as the ligand concentration increases the amount of unbound ligand-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured using a spectrophotometer.
In general, homogeneous assay systems require both an instrument to read the result and calibration of the observed signal by separate tests with samples containing known concentrations of ligand. The development of homogeneous assays has dominated competitive assay research and has resulted in several commercially available systems.
Heterogeneous, competitive ligand-receptor assays require a separation of bound labeled ligand or receptor from the free labeled ligand or receptor and a measurement of either the bound or the free fraction. Methods for performing such assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009. U.S. Pat. Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232, 4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe devices and methods for ligand-receptor assays that develop colored responses for visual interpretation of the results.
In the case of a competitive immunoassay, a labelled ligand reagent is bound to a limited and known quantity of receptor reagent. After that reaction reaches equilibrium, the ligand to be detected is added to the mixture and competes with the labelled ligand for the limited number of receptor binding sites. The amount of labelled ligand reagent displaced, if any, in this second reaction indicates the quantity of the ligand to be detected present in the fluid sample.
Another approach, a non-competitive immunochromatographic assay, is described in U.S. Pat. Nos. 4,168,146 and 4,435,504. This assay provides a method for quantitatively determining the presence of a single analyte in a sample in a visually interpreted immunoassay. U.S. Pat. No. 5,089,391 describes a method for performing competitive ligand-receptor assays so as to be able to semiquantitatively or quantitatively determine
Carpenter Laura R.
Stahl Neil
Yancopoulos George D.
Kempler Gail M.
Kunz Gary L.
Landsman Robert S.
Palladino Linda O.
Regeneron Pharmaceuticals Inc.
LandOfFree
Methods of screening competitors of OB-R binding to... 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 screening competitors of OB-R binding to..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods of screening competitors of OB-R binding to... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2512791