WO1999040116A1 - Procede d'expression du recepteur de la thyreostimuline - Google Patents

Procede d'expression du recepteur de la thyreostimuline Download PDF

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Publication number
WO1999040116A1
WO1999040116A1 PCT/AU1999/000071 AU9900071W WO9940116A1 WO 1999040116 A1 WO1999040116 A1 WO 1999040116A1 AU 9900071 W AU9900071 W AU 9900071W WO 9940116 A1 WO9940116 A1 WO 9940116A1
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WO
WIPO (PCT)
Prior art keywords
tsh receptor
bacteriophage
receptor protein
human tsh
assay
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PCT/AU1999/000071
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English (en)
Inventor
Albert George Frauman
Bernard John Major
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The University Of Melbourne
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Publication date
Application filed by The University Of Melbourne filed Critical The University Of Melbourne
Priority to AU24043/99A priority Critical patent/AU2404399A/en
Publication of WO1999040116A1 publication Critical patent/WO1999040116A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to a process for the production of thyroid stimulating hormone (TSH) receptor protein, in particular a process for the expression of TSH receptor protein using a bacteriophage expression system.
  • the invention also relates to an assay for the detection or diagnosis of Graves' disease.
  • Graves' disease is the most common autoimmune disease affecting humans, occurring in up to 2-3% of the adult population. This condition is characterised by stimulating autoantibodies to the TSH receptor which enhance production of the thyroid hormones T 4 and T 3 . Ordinarily when the TSH receptor is stimulated by the pituitary derived TSH, T 4 and T 3 production causes negative feedback of TSH production. In Graves' disease, an exogenous stimulus to the TSH receptor is present (i.e. , autoantibodies) and there is no inhibitory feedback of thyroid hormones on this antibody production; hence, there is an unchecked overproduction of thyroid hormones.
  • TSH receptor a 7-transmembrane domain receptor
  • the TSH receptor is highly membrane bound and hydrophobic and therefore difficult to separate and solubilise from prokaryotic sources.
  • prokaryotic expression of the extracellular domain of the human TSH receptor (the most immunogenic region) has yielded small amounts of receptor which would be impractical to use for an immunodiagnostic assay; only 10-50/xg protein/L of E. Coli culture has been recovered in the most recent efforts by large research laboratories active in the field of autoimmune thyroid disease (Bobovnikova et al. Endocrinology 1997; 138: 588-593).
  • a method for the expression of human TSH receptor protein utilising a bacteriophage expression system.
  • human TSH receptor protein refers to the complete human TSH receptor protein, or to immunogenic fragments, derivatives or portions thereof. Human TSH receptor protein consists of three main domains, these being the extracellular, transmembrane and intracellular domains. The most immunogenic region is to be found in the extracellular domain. Accordingly the term "human TSH receptor protein” includes the extracellular domain, or immunogenic fragments, derivatives or portions thereof. The term also includes fusion proteins which include the human TSH receptor or an immunogenic fragment, derivative or portion thereof.
  • TSH receptor cDNA may be cloned (or ligated) into a bacteriophage expression system.
  • bacteriophage expression systems are known in the art, one such system is the SurfZAPTM vector as described in US Patent No. 5,128,256 to Stratagene.
  • a method for the production of human TSH receptor protein including the steps of: ligating human TSH receptor cDNA into a bacteriophage cloning vector; amplifying ligated cDNA in E. Coli; incorporating amplified cDNA from bacteriophage cloning vector into a helper phage; amplifying helper phage in E. Coli, and recovering helper phage.
  • bacteriophage cloning vector refers to an expression system including DNA from a bacteriophage source.
  • the DNA from the bacteriophage source may be DNA which allows bacteriophage transcription.
  • the bacteriophage cloning vector is a phagemid into which such bacteriophage DNA has been inserted.
  • phagemid refers to a bacterial plasmid in which bacteriophage DNA sequences exist and which enables exogenous DNA to be inserted.
  • the human TSH receptor cDNA inserted into the bacteriophage cloning vector may encode all of the human TSH receptor protein, or an immunogenic fragment, derivative or portion thereof.
  • the human TSH receptor cDNA may be ligated or inserted into the bacteriophage cloning vector using techniques known to the art.
  • TSH receptor DNA may be inserted in a plasmid vector following polymerase chain reaction (PCR) amplification of thyroid derived cDNA using oligonucleotide primers incorporating appropriate restriction sites matching the plasmid cloning site. Restriction digests may then be performed and purified digested cDNA ligated using standard techniques.
  • PCR polymerase chain reaction
  • Amplification of the cDNA encoding the human TSH receptor may be achieved using the standard technique of transforming E. Coli with the plasmid produced. Any suitable strain of E. Coli may be used.
  • the DNA may be recovered after amplification and the amplified insert may be excised from the cloning vector.
  • the excised product may be cloned into a helper phage comprising pure bacteriophage DNA. Further amplification of the helper phage, which may contain a phagemid library, in E.coli is then performed. This may be followed by infection with an additional helper phage to enable the production and release of recombinant phage particles displaying the TSH receptor protein on their surface.
  • the recovery of the bacteriophage may be achieved using techniques known to the art.
  • the presence of the cDNA encoding recombinant human TSH receptor protein or fragment thereof in the recovered, amplified helper phage can be confirmed using conventional PCR techniques using appropriate primers.
  • the protein expressed on the surface of the bacteriophage includes the recombinant human TSH receptor protein.
  • the presence of the TSH receptor protein can be confirmed by separating the bacteriophage or a protein extract thereof on an appropriate chromatography column.
  • the bacteriophage particles having recombinant human TSH receptor protein expressed on their surface are useful in assays for detecting Graves' disease, greater sensitivity can be achieved by isolating the recombinant human TSH receptor protein and using the purified protein in the assay.
  • partial or complete purification of the TSH receptor protein can be achieved by solubilising the protein, followed by fractionation using standard chromatographic procedures, such as ion exchange chromatography, gel filtration, hydrophobic interaction chromatography and affinity chromatography, amongst others.
  • the human TSH receptor cDNA may be first amplified in a bacteriophage and then transferred to an expression system using, for example, a helper phage.
  • the recombinant human TSH receptor protein product including the bacteriophage particles having TSH receptor protein expressed on their surface, fusion proteins such as fusion proteins comprising bacteriophage protein and TSH receptor protein, and purified human TSH receptor protein, when prepared in accordance with the methods described above, represents a further aspect of the present invention.
  • the bacteriophage or at least the final bacteriophage in which the product is expressed, is preferably of the lytic type, although lysogenic bacteriophage may also be used provided a suitable agent is employed to lyse the bacterial cells.
  • the recombinant protein can be immobilised by coating onto the surface of tubes or
  • the signal generated by these indicator molecules is a measure of the amount of antibody in the test sample.
  • 25 forms described above may be used in an assay, such as an ELISA assay, to enable Graves' disease to be diagnosed and/or monitored.
  • an assay such as an ELISA assay
  • recombinant TSH receptor protein in an assay for the diagnosis and/or monitoring of Graves' disease.
  • the recombinant human TSH receptor protein is prepared in accordance
  • the recombinant human TSH receptor protein may be in the form of protein expressed on the surface of a bacteriophage as described above, or may be a fusion protein incorporating human TSH receptor protein, or may be purified recombinant human TSH receptor protein.
  • This assay will allow, for the first time, a rapid and sensitive assay for the diagnosis and monitoring of Graves' disease.
  • Previous assays are expensive, time consuming and depend on radioactivity and the provision of thyroid membrane preparations or cells in culture from animal sources.
  • the assay according to the present invention alleviates these problems by being rapid and sensitive, obviating the need for reactivity and, importantly, uses recombinant protein only. This avoids the use of animal tissues.
  • the use of this assay will enable the "tracking" of immunoreactivity during Graves' disease treatment, in order to more accurately predict relapse or the development of neonatal Graves' disease.
  • Figure 1 shows a plot of absorbance (optical density measured at 450 nm) for Graves' disease and normal serum samples, reacting against the SI region of the TSH receptor expressed in a bacteriophage;
  • Figure 2 shows a plot of absorbance (optical density measured at 450nm) for Graves' disease and normal serum samples, reacting against the S2 region of the TSH receptor expressed in a bacteriophage;
  • Figure 3 shows a plot of absorbance (optical density measured at 450nm) for Graves' disease and normal serum samples, reacting against the S3 region of the TSH receptor expressed in a bacteriophage; - 7
  • TSH receptor RNA extracted from normal thyroid using conventional methods is reverse transcribed into cDNA and three continuous regions of the extracellular domain of the TSH receptor are derived, using TSH receptor specific oligonucleotide primers, by PCR. These regions have been termed SI, S2 and S3 and correspond to, in sequence (5 '-3'), to 351, 420 and 670 nucleotides of the extracellular domain of the TSH receptor respectively.
  • TSH receptor DNA to be inserted in the vector is produced by PCR of thyroid cDNA using primers specific for the TSH receptor and compatible with the SurfZAPTM vector (Stratagene) ie. they contain the appropriate restriction enzyme sites, are in frame after ligation and the sequence for the missing portion of the vector at the ligation site is included.
  • the digested DNA is then dephosphorylated by incubating at 37 °C for 30 minutes in a total volume of 15 ⁇ l containing 1 x OPA buffer and 0.1 units calf intestine alkaline phosphatase (CIAP; Pharmacia Biotech).
  • the CIAP is inactivated by incubating at 70°C for 10 minutes.
  • the insert DNA fragments are now ready to be ligated to the Tag sequence.
  • Primers coding for a tag protein (I-spy; AMRAD Pharmacia) are then ligated to the insert DNA to aid the identification of expressed TSH receptor protein, using the following method.
  • the I-spy sequence codes for a 6 amino acid peptide to which a monoclonal antibody exists.
  • I-spy primers are designed to be flanked with Spe I sticky ends.
  • a single base is substituted (5'-3'; A ⁇ T) in - 8
  • I-spy primers are first hybridised to give double stranded DNA.
  • the primers are dissolved in distilled water and their concentration determined spectrophotometrically.
  • the forward and backward primer are mixed at a ratio of 1: 1 and made up to a final concentration of 50 pmol of DNA/ ⁇ l.
  • the primers are then heated to 90 °C for 20 minutes.
  • the heating block is then switched off and the primers allowed to cool slowly to allow annealing.
  • the primers should now be double stranded and are ready for ligation.
  • the Tag sequences are ligated to the TSH receptor insert fragments as follows: Approximately 300 ng of insert DNA is incubated with approximately 450 ng annealed I-spy primers in a total volume of 10 ⁇ l containing 30 mM Tris-HCl pH7.8, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM ATP and 3 units of T 4 DNA ligase (Promega). Ligation takes place at 4°C overnight followed by heat inactivation at 75 °C for 20 minutes.
  • T 4 PNK Polynucleotide Kinase
  • the vector supplied in the SurfZAPTM kit is in the form of Not / and Spel digested dephosphorylated bacteriophage ⁇ vector arms. Ligation takes place at 4°C overnight in a total volume of 5 ⁇ l.
  • the reaction mixture contains 1 ⁇ l of vector arms with 350-500 ng of insert D ⁇ A, 0.5 ⁇ g of the supplied test insert (a human Fab fragment that binds to tetanus toxoid) or 1 ⁇ l of distilled water in 50 mM Tris-HCl pH7.5, 7 mM MgCl 2 , 1 mM DTT, 1 mM ATP and 2 units of T 4 D ⁇ A ligase (Stratagene), with the glycerol content not exceeding 5% in the final volume.
  • Packaging extracts supplied by Stratagene are warmed between fingers until thawing commences. 4 ⁇ l of ligation mixture is added immediately and stirred gently with the pipette tip, then briefly vortexed. The packaging mixture is incubated at room temperature for 105 minutes then 500 ⁇ l of SM buffer (0.1 M ⁇ aCl, 14.5 mM MgSO 4 50 mM Tris-HCl pH7.5, and 0.01 % gelatin) and 20 ⁇ l of chloroform are added and mixed gently. The samples are centrifijged briefly and the supernatant, containing the packaged ⁇ bacteriophage, transferred to a fresh tube.
  • SM buffer 0.1 M ⁇ aCl, 14.5 mM MgSO 4 50 mM Tris-HCl pH7.5, and 0.01 % gelatin
  • Escherichia coli strain XLl-Blue is prepared for ⁇ bacteriophage growth and titre estimation as follows: The XLl-Blue are revived by streaking a splinter from a glycerol stock onto an LB- tetracycline plate.
  • a liquid overnight culture is set up as follows: A single colony from the LB- tetracycline plate is used to inoculate 10 ml of LB broth supplemented with 0.2% w/v maltose and 10 mM MgSO 4 . The culture is incubated at 37 °C with vigorous shaking overnight then centrifuged at 1000 g for 10 minutes at 4°C. The pellet is then resuspended in 10 mM
  • Serial dilutions of the ⁇ bacteriophage solution are prepared in SM buffer. 1-10 ⁇ l of the dilutions are mixed with 200 ⁇ l of the prepared host cells which have been diluted to an ODg oo of 0.5 with 10 ml MgSO 4 . The phage and bacteria mix are incubated at 37 °C for
  • volume plated in microliters where the volume plated in microliters refers to the volume of the phage solution added to the cells.
  • 0.5 ml of prepared host cells are then used to inoculate 25 ml of LB broth supplemented with 0.2% w/v maltose and 10 mM MgSO 4 .
  • the cells are grown at 37 °C with vigorous shaking until an OD 600 of between 0.2-0.3 is achieved and the culture is then centrifuged at 1000 g for 10 minutes at 4°C and the pellet resuspended to an OD 600 of 0.5 with 10 mM MgSO 4 .
  • the entire packaging mixture is added to 1 ml of the freshly prepared
  • the supernatant containing the amplified ⁇ bacteriophage library is transferred to a fresh sterile tube, chloroform to a final volume of 0.1 % v/v is added and the supernatant is stored at 4°C. The supernatant is titred as above.
  • XLl-Blue cells as prepared are mixed with ⁇ bacteriophage at a ratio of 10: 1 respectively (assuming an OD 600 of 0.5 equals a cell concentration of 4 x 10 9 cells/ml).
  • ExAssistTM helper phage is then added to the mixture at a ratio of 1 helper phage for every cell. The mixture is then incubated at 37°C with gentle agitation for 15 minutes. 20 ml of LB broth supplemented with 0.2% w/v maltose and 10 mM MgSO 4 is added and incubated at 37 °C with gentle agitation for 3 hours.
  • the excised phagemids are harvested by heating the culture at 70 °C for 20 minutes, which lyses the ⁇ particles and the XLl-Blue cells, and the centrifuged at 2500 g for 10 minutes at 4°C. The supernatant containing the excised phagemids is transferred to a fresh tube and stored at 4°C.
  • Serial dilutions of the excised phagemids are prepared in TE buffer. 1-10 ⁇ l of the dilutions are mixed with 200 ⁇ l of prepared SOLRTM cells with an OD 600 of 1.0 in 10 mM MgSO 4 . The phage and bacteria mix are incubated at 37 °C for 15 minutes then spread with a sterile glass spreader onto LB plates containing 50 ⁇ g/ml ampicillin. The plates are incubated at 37 °C overnight before being inspected for the formation of colonies. The number of colony forming units per ml (cm/ml) are calculated as follows:
  • volume plated in microliters where the volume plated in microliters refers to the volume of the phage solution added to the 11
  • 0.5 ml of prepared SOLRTM cells are used to inoculate 10 ml of LB broth containing 50 ⁇ g/ml Kanamycin. The cells are grown at 37 °C with vigorous shaking until an OD 600 of 0.5 is achieved. The culture is centrifuged at 1000 g for 10 minutes at 4°C and the pellet resuspended to an OD 600 of 1.0 with 10 mM MgSO 4 . 1 ml of the freshly prepared SOLRTM cells are incubated with 1 ml of the excision supernatant and incubated at 37 °C for 15 minutes.
  • the VCSM13 helper phage (Stratagene) was titred using the same method as used for the excised phagemids with the exception of LB-50 ⁇ g/ml Kanamycin plates being used instead of LB-ampicillin plates.
  • a 1 ml aliquot of the SOLRTM cells containing the phagemid library are used to inoculate a 10 ml LB broth containing 50 ⁇ g/ml Kanamycin and 100 ⁇ g/ml Carbenicillin. The cells are grown at 37 °C with vigorous shaking for 1-2 hours.
  • the culture is centrifuged at 1000 g for 10 minutes at 4°C and the pellet resuspended in 1 ml of LB broth plus 50 ⁇ g/ml Kanamycin and 100 ⁇ g/ml Carbenicillin.
  • VCSM13 helper phage are added at a ratio of 1: 1 with the cells (assuming that an OD ⁇ of 1.0 equals 8xl0 8 cells), and incubated at 37°C with gentle agitation for 15 minutes. 10 ml of LB broth plus 50 ⁇ g/ml Kanamycin and 100 ⁇ g/ml Carbenicillin is then added and incubated at 30 °C with gentle agitation overnight.
  • the culture is centrifuged at 2500 g for 30 minutes at 4°C and the pellet discarded.
  • the Supernatant containing the phage is transferred to a fresh tube and 0.2 volumes of PEG solution are added, vortexed and incubated on ice for 4 hours.
  • the solution is centrifuged at 10,000 g for 50 minutes and the pellet resuspended in 1 ml of TE buffer.
  • the dissolved pellet is then centrifuged in a microfuge at 10,000 g for 5 minutes at room temperature, to remove any insoluble matter and the supernatant transferred to a fresh tube, 0.2 volumes of PEG solution added and incubated at 4°C overnight.
  • the sample is then centrifuged in a microfuge for 10 minutes at room temperature and the pellet consisting of the purified phage - 12
  • filamentous phage are titred using the same method as above.
  • LB-ampicillin Plates containing a 3 ml top layer consisting of 0.266 mg/ml X-Gal, 0.22g mg/ml IPTG and 30 ⁇ g/ml ampicillin are also used to check for disruption of the ⁇ galactosidase gene complimentation by the inserted DNA.
  • the primers used are either the original primers used to create the insert DNA or additional TSH receptor specific primers which span a section of the TSH receptor internal to the original primers.
  • TSH receptor cDNA inserts are performed by PCR using TSH receptor oligonucleotide primers and also by restriction digests using Notl/Spel cuts; these have confirmed the presence of the TSH receptor inserts.
  • An ELISA assay consisting of bacteriophage at a density of 10 9 cfu (colony- forming units)/ml plated on the bottom of 96 well ELISA plates is used to detect reactivity of Graves' disease sera vs. non-Graves' disease sera. This ELISA has been performed as follows:
  • Diluted serum is applied to the bottom of the bacteriophage-coated plates (which contain the TSH receptor inserts or a control wild-type bacteriophage which does not contain the insert).
  • the serum is incubated then washed and a second antibody (a sheep antihuman IgG [ ⁇ -chain] antibody) is incubated in the ELISA wells. Any IgG from the human sera bound to the TSH receptor is thus bound by this second antibody.
  • the second antibody is conjugated to an enyzme, horseradish peroxidase (HRPO), which catalyses the chromogenic reaction of a specific substrate, TMB. This chromogenic reaction is then read on an ELISA plate reader at 450 nm and arbitrary units of absorbance are plotted.
  • HRPO horseradish peroxidase
  • Bacteriophage containing the TSH receptor inserts may be treated to selectively purify off the TSH receptor moiety which would then be used as an ELISA substrate.
  • the phage is solubilised in a non-ionic detergent (Tween or n-octyl glucopyranoside) and applied in dilute phosphate buffer pH7 to ion-exchange columns (DEAE-cellulose). Fractions are eluted with increasing concentrations of sodium chloride (up to 2M). Fractions containing the TSH receptor are identified by running them on an SDS-PAGE gel and immunoblotting with Graves' sera.
  • This additional step would be expected to enhance the sensitivity of the assay by a more concentrated preparation of TSH receptor protein and by elimination of irrelevant bacteriophage proteins which might otherwise interfere with the assay.

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Abstract

L'invention porte sur un procédé d'expression de la protéine réceptrice de la thyréostimuline, au moyen d'un système d'expression bactériophage, et sur un nouveau dosage pour le diagnostic et/ou la surveillance de la maladie de Graves.
PCT/AU1999/000071 1998-02-03 1999-02-03 Procede d'expression du recepteur de la thyreostimuline WO1999040116A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU24043/99A AU2404399A (en) 1998-02-03 1999-02-03 Process for expression of thyroid stimulating hormone receptor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPP1622A AUPP162298A0 (en) 1998-02-03 1998-02-03 Process for expression of thyroid stimulating hormone receptor
AUPP1622 1998-02-03

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WO1999040116A1 true WO1999040116A1 (fr) 1999-08-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004098652A1 (fr) * 2003-05-05 2004-11-18 Universita' Degli Studi Di Roma La Sapienza Hormone stimulant la thyroide (tsh) radiomarquee et son utilisation pour le diagnostic et le traitement des tumeurs differenciees de la thyroide

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CLINICA CHIMICA ACTA, Volume 263(1), 1997, NAKAI et al., "Recombinant Human TSH Receptor Expressed in E.Coli", pages 15-23. *
ENDOCRINOLOGY, Volume 134, No. 2, February 1994, SEETHARAMAIAH et al., "A Recombinant Extracellular Domain of the Thyrotropin (TSH) Receptor Binds TSH in the Absence of Membranes", pages 549-554. *
ENDOCRINOLOGY, Volume 138, No. 2, February 1997, BOBOVNIKOVA et al., "Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain", pages 588-593. *
EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES, Volume 105(5), 1997, MINICH et al., "Expression of a Functional Tagged Human Thyrotropin Receptor in HeLa Cells Using Recombinant Vaccinia Virus", pages 282-290. *
JOURNAL OF AUTOIMMUNITY, Volume 6, No. 3, June 1993, McINTOSH et al., "Use of Epstein-Barr Virus-Based Vectors for Expression of Thyroid Auto-Antigens in Human B-Lymphoblastoid Cell Lines", pages 353-365. *
JOURNAL OF MOLECULAR ENDOCRINOLOGY, Volume 10(2), 1993, HUANG et al., "The Thyrotropin Hormone Receptor of Graves' Disease: Overexpression of the Extracellular Domain in Insect Cells Using Recombinant Baculovirus, Immunoaffinity Purification and Analysis of Autoantibody Binding", pages 127-142. *
TRENDS IN ENDOCRINOLOGY AND METABOLISM, Volume 2, No. 4, VASSART et al., "Molecular Genetics of the Thyrotropin Receptor", pages 151-156. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004098652A1 (fr) * 2003-05-05 2004-11-18 Universita' Degli Studi Di Roma La Sapienza Hormone stimulant la thyroide (tsh) radiomarquee et son utilisation pour le diagnostic et le traitement des tumeurs differenciees de la thyroide

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