Antibodies to vertebrate delta proteins and fragments

An antibody, which binds a human Delta protein, which human Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:

• 14 and SEQ ID NO:26, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μ/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μ/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• which antibody does not bind a Drosophila Delta protein.

A composition comprising an antibody which binds to a vertebrate Delta protein;

• and a pharmaceutically acceptable carrier, which vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• and which antibody does not bind a Drosophila Delta protein.

A composition comprising a fragment of an antibody to a vertebrate Delta protein containing the binding domain of the antibody;

• and a pharmaceutically acceptable carrier, which vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• and which antibody does not bind a Drosophila Delta protein.

A method of making an antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a host animal, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• such that an antibody to said Delta protein is produced by said host animal;
• and
• (b) recovering the antibody.

A method of making an antibody comprising:

• (a) administering an immunogenic amount of a fragment of a vertebrate Delta protein to a host animal, in which the fragment comprises a domain of the protein selected from the group consisting of the extracellular domain, DSL domain, domain amino-terminal to the DSL domain, epidermal growth factor-like repeat 1, epidermal growth factor-like repeat 2, and intracellular domain, in which the vertebrate Delta protein comprises an amino acid sequence encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• such that an antibody to said fragment is produced by said host animal;
• and
• (b) recovering the antibody.

A method of making an antibody comprising:

• (a) administering an immunogenic amount of a protein comprising a fragment of a vertebrate Delta protein to a host animal, in which the fragment comprises a domain of the protein selected from the group consisting of the extracellular domain, DSL domain, domain amino-terminal to the DSL domain, epidermal growth factor-like repeat 1, epidermal growth factor-like repeat 2, and intracellular domain, in which the vertebrate Delta protein comprises an amino acid sequence encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.0 1% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• such that an antibody to said Delta fragment is produced by said host animal;
• and
• (b) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a mouse, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• (b) recovering spleen cells from said mouse;
• (c) fusing the recovered spleen cells with a cell of a mouse myeloma to generate hybridomas;
• (d) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (e) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) fusing a spleen cell from a mouse immunized with an immunogenic amount of a vertebrate Delta protein with a cell of a mouse myeloma to generate hybridomas, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ 1D NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• (b) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (c) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a host animal, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• (b) recovering lymphocytes from said host animal;
• (c) fusing the recovered lymphocytes with a cell of a myeloma, plastocytoma or lymphoblastoid cell line to generate hybridomas;
• (d) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (e) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) fusing a lymphocyte from a host animal immunized with an immunogenic amount of a vertebrate Delta protein with a cell of a myeloma, plastocytoma or lymphoblastoid cell line to generate hybridomas, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:
• 14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• (b) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (c) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a host animal, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringency conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• (b) recovering lymphocytes from said host animal;
• (c) immortalizing the recovered lymphocytes with Epstein-Barr virus to generate immortalized cells;
• (d) screening to select an immortalized cell producing antibody to said vertebrate Delta protein;
• and
• (e) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) immortalizing a lymphocyte from a host animal immunized with an immunogenic amount of a vertebrate Delta protein with Epstein-Barr virus to generate immortalized cells, in which the vertebrate Delta protein is encoded by a first nucleic acid that hybridizes under high stringency conditions to a second nucleic acid, or its complement, the nucleotide sequence of the second nucleic acid being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:24, said high stringehcy conditions comprising pretreatment for 8 hours to overnight in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA;
• hybridization for 48 hours at 68° C. in a solution containing 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon sperm DNA;
• washing for 1 hour at 37° C. in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA;
• and a second washing for 45 minutes at 50° C. in a solution containing 0.1×SSC;
• (b) screening to select an inunortalized cell producing antibody to said vertebrate Delta protein;
• and
• (c) recovering the antibody.

A method of making an antibody comprising:

• (a) administering an immunogenic amount of a fragment of a vertebrate Delta protein to a host animal, such that an antibody to said fragment is produced by said host animal;
• in which the fragment comprises a domain of the vertebrate Delta protein selected from the group consisting of the extracellular domain, DSL domain, domain amino-terminal to the DSL domain, epidermal growth factor-like repeat 1, epidermal growth factor-like repeat 2, and intracellular domain;
• in which the vertebrate Delta protein comprises SEQ ID NO:65;
• and
• (b) recovering the antibody.

A method of making an antibody comprising:

• (a) administering an immunogenic amount of a protein comprising a fragment of a vertebrate Delta protein to a host animal, such that an antibody to said Delta fragment is produced by said host animal;
• in which the fragment comprises a domain of the vertebrate Delta protein selected from the group consisting of the extracellular domain, DSL domain, domain amino-terminal to the DSL domain, epidermal growth factor-like repeat 1, epidermal growth factor-like repeat 2, and intracellular domain;
• in which the vertebrate Delta protein comprises SEQ ID NO:65;
• and
• (b) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a mouse, which vertebrate Delta protein comprises SEQ ID NO:65;
• (b) recovering spleen cells from said mouse;
• (c) fusing the recovered spleen cells with a cell of a mouse myeloma to generate hybridomas;
• (d) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (e) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) fusing a spleen cell from a mouse immunized with an immunogenic amount of a vertebrate Delta protein with a cell of a mouse myeloma to generate hybridomas, which vertebrate Delta protein comprises SEQ ID NO:65;
• (b) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (c) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a host animal, in which the vertebrate Delta protein comprises SEQ ID NO:65;
• (b) recovering lymphocytes from said host animal;
• (c) fusing the recovered lymphocytes with a cell of a myeloma, plastocytoma or lymphoblastoid cell line to generate hybridomas;
• (d) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (e) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) fusing a lymphocyte from a host animal immunized with an immunogenic amount of a vertebrate Delta protein with a cell of a myeloma, plastocytoma or lymphoblastoid cell line to generate hybridomas, which vertebrate Delta protein comprises SEQ ID NO:65;
• (b) screening to select a hybridoma producing antibody to said vertebrate Delta protein;
• and
• (c) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) administering an immunogenic amount of a vertebrate Delta protein to a host animal, which vertebrate Delta protein comprises SEQ ID NO:65;
• (b) recovering lymphocytes from said host animal;
• (c) immortalizing the recovered lymphocytes with Epstein-Barr virus to generate immortalized cells;
• (d) screening to select an immortalized cell producing antibody to said vertebrate Delta protein;
• and
• (e) recovering the antibody.

A method of making a monoclonal antibody comprising:

• (a) immortalizing a lymphocyte from a host animal immunized with an immunogenic amount of a vertebrate Delta protein with Epstein-Barr virus to generate immortalized cells, which vertebrate Delta protein comprises SEQ ID NO:65;
• (b) screening to select an immortalized cell producing antibody to said vertebrate Delta protein;
• and
• (c) recovering the antibody.