Sulfonated sclerostin, antibodies, epitopes and methods for identification and use therefor
Provided are antibodies that bind to: a sulfonated epitope of the protein Sclerostin, to Sclerostin portions comprising a sulfonated amino acid and to dimerized forms of Sclerostin. Further provided are compositions and peptides comprising a sulfonated epitope of sclerostin. Also provided by this invention are methods for production of such antibodies, both active and passive, and methods for identifying antibodies specific for sulfonation sites in Sclerostin and other antibodies which discriminate between sulfonated and unsulfonated forms of sclerostin. Physical and virtual screening processes are provided in this invention for identifying compounds which disrupt or inhibit sulfonation and the interaction between Sclerostin and binding partners. The antibodies and compositions of the present invention are useful in diagnostic and therapeutic applications directed to Sclerostin-related disorders.
1. A method for production of a monoclonal antibody specific for a sulfonation site in Sclerostin, comprising the steps of:
immunizing an animal with a synthetic peptide derived from the Sclerostin amino acid sequence, wherein said peptide comprises a sulfonation site in said Sclerostin that comprises a tyrosine corresponding to Tyr43 or Tyr213 of human Sclerostin;
isolating antibody producing cells from said immunized animal;
fusing the isolated antibody producing cells with myeloma cells to form antibody producing hybridoma cells; and
screening the hybridoma cells to identify hybridoma cells that produce antibodies that discriminate between sulfonated and unsulfonated forms of said sulfonation site of Sclerostin.
2. The method of claim 1 , wherein said tyrosine is sulfonated in the synthetic peptide.
3. The method of claim 1 , wherein said tyrosine corresponds to Tyr43 of human Sclerostin.
4. The method of claim 3 , wherein said tyrosine is sulfonated in the synthetic peptide.
5. The method of claim 1 , wherein the synthetic peptide is selected from the group consisting of:
(a) ELGEYPEPPPELE (SEQ ID NO:8), wherein tyrosine is sulfonated;
(b) ELGEYPEPPPELE (SEQ ID NO:8), wherein tyrosine is not sulfonated;
(c) KANQAELENAY (SEQ ID NO:7), wherein tyrosine is sulfonated; and
(d) KANQAELENAY (SEQ ID NO:7), wherein tyrosine is not sulfonated.
6. The method of claim 5 , wherein the peptide is ELGEYPEPPPELE (SEQ ID NO:8), wherein tyrosine is sulfonated.