Novel human beta-2 integrin alpha subunit

a human beta2 and alpha subunit technology, applied in the field of new human beta2 integrin alpha subunits, can solve the problem of inability to define the correlation between human beta2 integrin subunits and those identified in other species

Inactive Publication Date: 2006-12-14
ICOS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017] Assays to identify αd binding molecules are also provided, including in vitro assays such as immobilized ligand binding assays, solution binding assays, and scintillation proximity assays, as well as cell based assays such as di-hybrid screening assays, split hybrid screening assays, and the like. Cell based assays provide for a phenotypic change in a host cell as a result of specific binding interaction or disruption of a specific binding interaction, thereby permitting indirect quantitation or measurement of some specific binding interaction.
[0018] In vitro assays for identifying antibodies or other compounds that modulate the activity of αd may involve, for example, immobilizing αd or a natural ligand to which αd binds, detectably labelling the nonimmobilized binding partner, incubating the binding partners together and determining the effect of a test compound on the amount of label bound wherein a reduction in the label bound in the presence of the test compound compared to the amount of label bound in the absence of the test compound indicates that the test agent is an inhibitor of αd binding.

Problems solved by technology

The absolute molecular weights of presumed homologs from other species have been shown to vary significantly [see, e.g., Danilenko et al., Tissue Antigens 40:13-21 (1992)], and in the absence of sequence information, a definitive correlation between human integrin subunits and those identified in other species has not been possible.

Method used

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  • Novel human beta-2 integrin alpha subunit
  • Novel human beta-2 integrin alpha subunit
  • Novel human beta-2 integrin alpha subunit

Examples

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Effect test

example 1

Attempt to Detect a Human Homolog of Canine αTM1

[0055] The monoclonal antibody Ca11.8H2 [Moore, et al., supra] specific for canine αTM1 was tested for cross-reactivity on human peripheral blood leukocytes in an attempt to identify a human homolog of canine αTM1. Cell preparations (typically 1×106 cells) were incubated with undiluted hybridoma supernatant or a purified mouse IgG-negative control antibody (10 μg / ml) on ice in the presence of 0.1% sodium azide. Monoclonal antibody binding was detected by subsequent incubation with FITC-conjugated horse anti-mouse IgG (Vector Laboratories, Burlingame, Calif.) at 6 μg / ml. Stained cells were fixed with 2% w / v paraformaldehyde in phosphate buffered saline (PBS) and were analyzed with a Facstar Plus fluorescence-activated cell sorter (Becton Dickinson, Mountain View, Calif.). Typically, 10,000 cells were analyzed using logarithmic amplification for fluorescence intensity.

[0056] The results indicated that Ca11.8H2 did not cross-react with ...

example 2

Affinity Purification of Canine αTM1 for N-Terminal Sequencing

[0058] Canine αTM1 was affinity purified in order to determine N-terminal amino acid sequences for oligonucleotide probe / primer design. Briefly, anti-αTM1 monoclonal antibody Ca11.8H2 was coupled to Affigel® 10 chromatographic resin (BioRad, Hercules, Calif.) and protein was isolated by specific antibody-protein interaction. Antibody was conjugated to the resin, according to the BioRad suggested protocol, at a concentration of approximately 5 mg antibody per ml of resin. Following the conjugation reaction, excess antibody was removed and the resin blocked with three volumes of 0.1 M ethanolamine. The resin was then washed with thirty column volumes of phosphate buffered saline (PBS).

[0059] Twenty-five grams of a single dog spleen were homogenized in 250 ml of buffer containing 0.32 M sucrose in 25 mM Tris-HCl, Ph 8.0, with protease inhibitors. Nuclei and cellular debris were pelleted with centrifugation at 1000 g for 15...

example 3

Large Scale Affinity Purification of Canine αTM1 for Internal Sequencing

[0069] In order to provide additional amino acid sequence for primer design, canine αTM1 was purified for internal sequencing. Three sections of frozen spleen (approximately 50 g each) and frozen cells from two partial spleens from adult dogs were used to generate protein for internal sequencing. Fifty grams of spleen were homogenized in 200-300 ml borate buffer with a Waring blender. The homogenized material was diluted with 1 volume of buffer containing 4% NP-40, and the mixture then gently agitated for at least one hour. The resulting lysate was cleared of large debris by centrifugation at 2000 g for 20 min, and then filtered through either a Corning (Corning, N.Y.) prefilter or a Corning 0.8 micron filter. The lysate was further clarified by filtration through the Corning 0.4 micron filter system.

[0070] Splenic lysate and the antibody-conjugated Affigel® 10 resin described in Example 2 were combined at a 1...

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Abstract

Methods to inhibit inflammation and macrophage infiltration following spinal cord injury are disclosed along with methods to modulate TNFα release from cells expressing αd are disclosed.

Description

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 08 / 943,363 filed Oct. 3, 1997, which is pending, which a continuation-in-part of U.S. patent application Ser. No. 08 / 605,672, filed Feb. 22, 1996, which issued as U.S. Pat. No. 5,817,515 on Oct. 6, 1998, which is a continuation-in-part of U.S. application Ser. No. 08 / 362,652, filed Dec. 21, 1994, which issued as U.S. Pat. No. 5,766,850 on Jun. 16, 1998, which is a continuation-in-part of U.S. application Ser. No. 08 / 286,889, filed Aug. 5, 1994, which issued as U.S. Pat. No. 5,470,953 on Nov. 28, 1995, which in turn is a continuation-in-part of U.S. application Ser. No. 08 / 173,497, filed Dec. 23, 1993, which issued as U.S. Pat. No. 5,437,958 on Aug. 1, 1995.BACKGROUND OF THE INVENTION [0002] The integrins are a class of membrane-associated molecules which actively participate in cellular adhesion. Integrins are transmembrane heterodimers comprising an α subunit in noncovalent association with a β su...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395A61K38/00C07K14/705C07K16/28C12Q1/68G01N33/68
CPCA01K2217/05A61K38/00A61K2039/505C07K14/70553G01N2333/70553C07K2319/00C12Q1/6818G01N33/6893C07K16/2845
Inventor GALLATIN, W.VAN DER VIEREN, MONICA
Owner ICOS CORP
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