Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1991-10-15
2001-05-08
Venkat, Jyothsna (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007200, C435S007210, C435S069100, C436S500000, C436S518000, C530S350000, C530S854000, C530S388220, C530S387900, C536S023500
Reexamination Certificate
active
06228597
ABSTRACT:
The invention relates to polypeptides having thyrotropin-receptor activity, to nucleic acids coding for such polypeptides, to antibodies to these polypeptides and to the use of the polypeptides and antibodies in assay methods.
The literature references indicated by numbers in parentheses in this specification are listed in the form of a bibliography at the end of the description.
Pituitary glycoproteins (Luteinizing hormone, LH; follicle stimulating hormone, FHS; and thyroid stimulating hormone or thyrotropin, TSH) form a family of closely related hormones.
The pituitary hormone thyrotropin (TSH) is the main physiological agent regulating the thyroid gland. It stimulates the function and the proliferation of thyrocytes and induces the expression of differentiation (1). Most of its effects are mediated by cyclic AMP (cAMP) (1). As the other pituitary and placental glycoprotein hormones (FSH, LH, CG), TSH is a heterodimer. All these hormones share an identical alpha subunit; the beta subunit, despite sequence similarity, is specific for each (2). The activated TSH, FSH and LH-CG receptors stimulate adenylyl cyclase in their target cells via mechanisms mediated by the G protein Gs (3). In man, the TSH receptor may be the target of autoimmune reactions leading to hyper- or hypo-stimulation of the thyroid gland by autoantibodies in Grave's disease and in idiopathic myxoedema, respectively (4).
A prerequisite to studies of such diseases and to the elucidation of receptor structure and function is the availability of receptor preparations, particularly human, at a reasonable cost and in relative abundance.
To date, particulate membrane preparations and detergent-solubilised thyroid membranes, often of porcine or bovine origin (4) have been used in such studies. Human receptor preparations are not only costly but are also difficult to reproduce identically. Furthermore, the known preparations cannot be considered to be “purified” receptors; they are enriched with respect to their receptor content but do not allow purification of the receptor to a degree which would enable a partial sequence analysis, and hence its cloning. These receptor preparations have never allowed characterisation of the entity responsible for the TSH-binding activity.
Cloning and expression of the related LH-CG receptor has recently been achieved. A cDNA for the rat LH-CG receptor was isolated with use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein (15). Variants of the porcine LH-CG receptor were cloned by screening a &lgr;gt11 library with cDNA probes isolated with monoclonal antibodies (16).
Attempts have been made to clone the TSH receptor (6) using a method which exploits the sequence similarity displayed by all known G-protein coupled receptors. A pair of oligonucleotide primers corresponding to transmembrane segments III and VI were used on cDNA from thyroid tissue under conditions allowing amplification of the primed sequences by the polymerase chain reaction. The method did not allow cloning of the TSH receptor but led instead to the cloning of four new members of the G-protein coupled receptor family.
The difficulties encountered in purifying and in cloning the TSH receptor are thought to be due to its extra-ordinary low abundance even in thyroid cells.
The present inventors have succeeded in cloning the TSH receptor and variants thereof, firstly by applying the technique described in (6) but with different sets of primers, and with human genomic DNA as the template, rather than cDNA and secondly by use of a selected sequence amplified by this technique as a probe.
REFERENCES:
patent: 4448764 (1984-05-01), Smith et al.
patent: 5144007 (1992-09-01), Pfahl
patent: 5614363 (1997-03-01), Cone
Nagayama et al., Mol. Endo. 6(2): 145-155, 1992.
Chan et al., Endrocrinology 120 (suppl.) T16 (Abstract 32), 1987.
Chan et al., J. Biol. Chem. 264(7): 3651-3654, 1989.
Frazier-Seabrook et al., Abstract 1072, Endocrine Society, 1989.
Rudinger J. Caracteristics of the amino acids as components of a peptide hormone sequence. Chapter 1 in “Peptide Hormones,” edited by J. A.Parsons, University Park Press, Baltimore, pp. 1-7, 1976.*
Watson et al . The G-protein linked receptor facts book. Academic Press, San Diego p. 5, 1994.*
Costa et al. Immunohistochemiustry on whole mount preparations. Chapter 14 in “Immunohistochemistry” edited by A.C. Cuello John Wiley and Sons, New York, pp. 373-393, 1983.*
Harlow et al. Labeling of antibodies with iodine in “Antibodies a laboratory manual” Cold Sping Harbor Press, pp. 324-339, 1988.*
Koizumi et al Endocrinology 110 #4pp 1381-1391 (1982).*
Valente et al PNAS 79 pp 6680-6684 (1984).*
Massart et al Clin.Chem. 32/7, 1332-1335(1986).*
McFarland et al Science 245 pp 494-499 (1989).*
Frazier-Seabrook et al, Program 64thMeeting of American Thyroid Ass. Abstract T-51(1989).*
Bedin et al Mol. and Cellular Endicronology 65 pp 135-144(1989).*
Webster et al Cell 54 pp 199-207 (1988).*
Rees Smith et al Meth. Enx. 74 pp 405-420 (1981).*
Nagayama et al Biochem. and Biophy. Res, Comm. 165 #3 pp 1184-1190 (1989).*
Yoshida et al Clinical Research 36 3(1988) 610A “Molecular Cloning of a cDNA Encoding a Human Thyrotropin (hTSH) Receptor: Identification of Receptor mRNA in Thyroid and IM9 Lymphocytes”.*
Young et al Proc.Natl. Acad. Sci. 80 (1983) pp. 1194-1198“Efficient isolation of genes by using antibody probes”.*
CattyAntibodiesvol. 1:A Practical Approach(1988) IRL Press Limited, pp 7-18.
DuMont Jacques
Libert Frederic
Parmentier Marc
Vassart Gilbert
B.R.A.H.M.S. Diagnostica GmbH
Pillsbury & Winthrop LLP
Ricigliano Joseph W.
Venkat Jyothsna
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