Assay for the detection of phosphorylated PTH
View Patent ↗Antibodies for a variant of intact parathyroid hormone (PTH) or its fragments which have at position 17 the amino acid serine which is phosphorylated [p(17) PTH] and methods of producing the same. The invention further includes various immunoassay methods which use the described antibody alone or in combination with other antibodies to determine the circulating levels of p(17) intact PTH (1-84) or fragments thereof in biological fluids. Such antibodies and immunoassays can be used in conjunction with other antibodies to detect all biologically active forms of hPTH present in biological fluids.
1. An isolated antibody or an antigen binding fragment thereof that specifically binds to human parathyroid hormone (SEQ ID NO. 1) having a phosphorylated serine amino acid at position 17.
2. The antibody or antibody fragment of claim 1 wherein the antibody or antigen binding fragment thereof specifically binds to human parathyroid hormone at an epitope consisting of amino acids 13-22 (SEQ ID NO. 2).
3. A method for producing antibodies useful in the determination of PTH levels in a biological sample, wherein the PTH has a phosphorylated serine amino acid at position 17 the method comprising the steps:
a. administering a first peptide antigen to a host animal to induce antibody production against said first peptide antigen in the host animal wherein said first peptide antigen comprises amino acids 13-22 (SEQ ID NO. 2) of human PTH (SEQ ID NO. 1), wherein the amino acid serine present at the seventeenth residue in said PTH and wherein said serine is phosphorylated;
b. monitoring antibody titer produced by the administration of said first peptide antigen to the host animal;
c. extracting antisera produced in said host animal;
d. subjecting the antisera extracted in step c) to affinity chromatography utilizing a second peptide antigen, wherein said second peptide antigen is a bioactive form of PTH selected from the group consisting of PTH (1-34) (SEQ ID NO. 4) and PTH (1-84) (SEQ ID NO. 1) wherein the bioactive form of PTH has a phosphorylated serine amino acid at position 17, to recover the antibodies from said antisera that have affinity for said second peptide antigen; and
e. subjecting said antibodies recovered in step d) to affinity chromatography utilizing a third peptide antigen, wherein said third peptide antigen is a non-phosphorylated form of bioactive PTH selected from the group consisting of PTH (1-34) (SEQ ID NO. 4) and PTH (1-84) (SEQ ID NO. 1), to recover at least one antibody that does not have affinity for said third peptide antigen.
4. The method of claim 3 wherein said first peptide antigen in step a) is coupled to a carrier protein.
5. An isolated antibody produced by the method of claim 3 .