Monoclonal antibodies against osteopontin
View Patent ↗The present invention relates to reagents and methods for the detection of osteopontin fragments and distinguishing them from each other and from the full-length osteopontin protein. The present invention also relates to assays for the determination of the presence of osteopontin fragments in samples obtained from subjects and, further, the correlation of osteopontin fragment levels fragment levels with disease detection, progression and prognosis.
1. A method of identifying fragments of osteopontin peptide as being C-terminal fragments of osteopontin peptide or N-terminal fragments of osteopontin peptide, the method comprising:
a) capturing the osteopontin fragments with a capture antibody which binds specifically to a first C-terminal epitope of osteopontin peptide in a first assay position and a first N-terminal epitope of osteopontin peptide in a second assay position;
b) detecting the presence of captured osteopontin fragments, if any, of the first and second assay positions with a second antibody specific for a second C-terminal epitope of osteopontin peptide in the first assay position and a second antibody specific for a second N-terminal epitope of osteopontin in the second assay position; and
c) determining i) if any peptides captured in the first assay position as detected by the detecting antibody are C-terminal fragments with a determination antibody which specifically binds a first or second N-terminal epitope of osteopontin, the lack of binding of the determination antibody in the first assay position in view of the binding of the detection antibody being indicative of the presence of C-terminal osteopontin fragments in the first assay position and; determining ii) if any captured osteopontin peptides in the second assay position as detected by the detection antibody are N-terminal fragments with a determination antibody which specifically binds a first or second C-terminal epitope of osteopontin, the lack of binding of the determination antibody in the second assay position in view of the binding of the detection antibody being indicative of the presence of N-terminal osteopontin fragments in the second assay position, as compared to a control sample comprising a full length osteopontin peptide.
2. The method of claim 1 , wherein said capture antibodies are selected from a group consisting of antibodies specific for epitopes located at amino acids 1-168 or amino acids 1-166 of the N-terminus of the osteopontin peptide and amino acids 167-314 or amino acids 169-314 of the C-terminus of the osteopontin peptide.
3. The method of claim 1 , wherein said determining antibodies are selected from a group consisting of antibodies specific for epitopes located at amino acids 1-168 or amino acids 1-166 of the N-terminus of the osteopontin peptide and amino acids 167-314 or amino acids 169-314 of the C-terminus of the osteopontin peptide.
4. The method of claim 1 , wherein said detecting antibodies are selected from a group consisting of antibodies specific for epitopes located at amino acids 1-168 or amino acids 1-166 of the N-terminus of the osteopontin peptide and amino acids 167-314 or amino acids 169-314 of the C-terminus of the osteopontin peptide.
5. The method of claim 1 , wherein the detection antibody is selected from the group consisting of antibodies specific for different N-terminal epitopes of osteopontin selected from the group consisting of 2C5 produced by a hybridoma deposited as ATCC Accession No. PTA-11447, 2H9 produced by a hybridoma deposited as ATCC Accession No. PTA-11446 and 2F10 produced by a hybridoma deposited as ATCC Accession No. PTA-11450 for the second assay position and specific for different C-terminal epitopes of osteopontin selected from the group consisting of 2E11 produced by a hybridoma deposited as ATCC Accession No. PTA-11449 and 1F11 produced by a hybridoma deposited as ATCC Accession No. PTA-11448 for the first assay position.
6. The method of claim 1 , wherein the capture antibody is selected from the group consisting of antibodies specific for different N-terminal epitopes of osteopontin selected from the group consisting of 2C5 produced by a hybridoma deposited as ATCC Accession No. PTA-11447, 2H9 produced by a hybridoma deposited as ATCC Accession No. PTA-11446 and 2F10 produced by a hybridoma deposited as ATCC Accession No. PTA-11450 for the second assay position and specific for different C-terminal epitopes of osteopontin selected from the group consisting of 2E11 produced by a hybridoma deposited as ATCC Accession No. PTA-11449 and 1F11 produced by a hybridoma deposited as ATCC Accession No. PTA-11448 for the first assay position.
7. The method of claim 1 , wherein the determining antibody is selected from the group consisting of antibodies specific for different N-terminal epitopes of osteopontin selected from the group consisting of 2C5 produced by a hybridoma deposited as ATCC Accession No. PTA-11447, 2H9 produced by a hybridoma deposited as ATCC Accession No. PTA-11446 and 2F10 produced by a hybridoma deposited as ATCC Accession No. PTA-11450 for the first assay position and specific for different C-terminal epitopes of osteopontin selected from the group consisting of 2E11 produced by a hybridoma deposited as ATCC Accession No. PTA-11449 and 1 F11 produced by a hybridoma deposited as ATCC Accession No. PTA-11448 for the second assay position.
8. The method of claim 1 , wherein said method is automated.
9. An antibody specific for an epitope on osteopontin, said antibody selected from a group consisting of 2C5, 2H9, 2F10, 2E11 and 1F11.