Antibodies that bind peptides from the hPTH sequence (1-37)

Details Antibodies that bind peptides from the hPTH sequence (1-37) Antibodies that bind peptides from the hPTH sequence (1-37)

1997-03-27

2000-02-29

Kemmerer, Elizabeth

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

436512, 5303871, 5303872, 5303879, 53038824, G01N 3343, C07K 1626

Patent

active

060307900

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to peptides from the sequence of hPTH(1-37), and the use of said peptides in the preparation of an agent for diagnosing biologically active hPTH.
Human parathyroid hormone (hPTH), a linear polypeptide having 84 amino acids, plays an important role in the regulation of the calcium metabolism. The metabolism of this hormone gives rise to a large number of C-terminal fragments, the biological functions of which have not yet been elucidated. The hPTH(1-37) has been established as a circulating N-terminal fragment (EP-A 0 349 545). This fragment has the full biological activity of the entire hormone. However, upon loss of the first amino acid, serine, the activity significantly decreases and is lost completely without the first two amino acids, serine and valine.
Serum levels in the range of 10.sup.-12 mol/l are measured for the intact hormone hPTH(1-84) and for the N-terminal fragment. Immunological measuring procedures are employed to determine such low concentrations. Here, the most valid results are provided by measuring procedures according to the double antibody or sandwich principle (e.g., the two-site radioimmunometric assay, IRMA, or the sandwich enzyme-linked immuno sorbent assay, Sandwich ELISA). For hPTH(1-84), such assays are commercially available. For the measurement of hPTH(1-34), an assay according to the double antibody principle is not known.
Here, two antibodies are required. In order to avoid mutual steric hindrance, they must be capable of recognizing antigen epitopes located at a sufficient distance from each other. When immunizing using the intact antigen, a heterogeneous mixture of various antibodies is obtained, which first must be subjected to an expensive purification in order to conduct a sandwich assay. According to theoretical calculations by B. A. Jameson and H. Wolf, The Antigenic Index: A Novel Algorithm for Predicting Antigenic Determinants, CABIOS 4, p. 181-186, 1988; it has been possible so far to detect a preferred sequence having immunogenic activity in the region of the amino acids 7-14 at the N-terminus. Immunization with N-terminal fragments according to established methods predominantly results in antibodies which, as has been described for hPTH(1-34) (J. Tampe, P. Brozio, H. E. Manneck, A. Mi.beta.bichler, E. Blind, K. B. Millers, H. SchmidtGayk, and F. P. Armbruster, Characterisation of Antibodies Against Human N-Terminal Parathyroid Hormone by Epitope Mapping; J. Immunoassay 13, p. 1-13, 1992), bind in the region of these amino acids. However, these antibodies are not capable of discriminating between biologically active and biologically inactive PTH(1-84) or fragments thereof lacking the first two amino acids serine and valine.
The technical problem which this invention is based upon is to provide peptides by means of which it is possible to eliminate the above-mentioned drawbacks in the diagnosis of biologically active hPTH.
Surprisingly, the technical problem described above is solved by means of the following peptides from the sequence of hPTH(1-37):


hPTH 1-10 SEQ I.D. NO.1 NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6 -Leu.sup.7 -Met.sup.8 -His.sup.9 -Asn.sup.10 -OH (1) hPTH 1-9 SEQ I.D. NO.2 NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6 -Leu.sup.7 -Met.sup.8 -His.sup.9 -OH (2) hPTH 1-8 SEQ I.D. NO.3 NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6 -Leu.sup.7 -Met.sup.8 -OH (3) hPTH 1-7 SEQ I.D. NO.4 NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6 -Leu.sup.7 -OH (4) hPTH 1-6 SEQ I.D. NO.5 NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6 -OH (5) hPTH 1-5 SEQ I.D. NO.6 NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -OH (6) hPTH 9-18 SEQ I.D. NO.7 NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13 -His.sup.14 -Leu.sup.15 -Asn.sup.16 -Ser.sup.17 -Met.sup.18 -OH (7) hPTH 10-18 SEQ I.D. NO.8 NH.sub.2 -Asn

REFERENCES:
patent: 4508828 (1985-04-01), Lindall et al.
Nussbaum et al. Chemical Abstracts 96(5), Abstract No. 29060, 1982.
Tampe et al. J. Immunoassay 13(1):1-13, 1992.
Daniel et al. Virology 202: 540-549, 1994.
Bowie et al. Science 247:1306-1310, 1990.
Ngo et al. The Protein Folding Problem and Tertiary Structure Prediction, Merz et al., eds., Birkhauser, Boston, pp. 492-495, 1994.

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