[go: up one dir, main page]

WO1998020354A2 - Controle de la reactivite d'auto-anticorps de la peroxydase thyroidienne - Google Patents

Controle de la reactivite d'auto-anticorps de la peroxydase thyroidienne Download PDF

Info

Publication number
WO1998020354A2
WO1998020354A2 PCT/GB1997/003014 GB9703014W WO9820354A2 WO 1998020354 A2 WO1998020354 A2 WO 1998020354A2 GB 9703014 W GB9703014 W GB 9703014W WO 9820354 A2 WO9820354 A2 WO 9820354A2
Authority
WO
WIPO (PCT)
Prior art keywords
tpo
modified
disease
carried out
abs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1997/003014
Other languages
English (en)
Other versions
WO1998020354A3 (fr
Inventor
Fiona Grennan Jones
Jadwiga Furmaniak
Bernard Rees Smith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
RSR Ltd
Original Assignee
RSR Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by RSR Ltd filed Critical RSR Ltd
Priority to JP52113998A priority Critical patent/JP2001503983A/ja
Priority to EP97910562A priority patent/EP0935756A2/fr
Publication of WO1998020354A2 publication Critical patent/WO1998020354A2/fr
Publication of WO1998020354A3 publication Critical patent/WO1998020354A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • the present invention relates to a method of monitoring the reactivity of thyroid peroxidase (TPO) autoantibodies (Abs) in order to distinguish between disease-related and disease-unrelated TPO Abs.
  • TPO thyroid peroxidase
  • Abs autoantibodies
  • thyroid microsomal antigen is thyroid peroxidase (TPO) -the enzyme involved in iodination of tyrosines in thyroglobulin i.e. formation of thyroid hormones.
  • TPO thyroid peroxidase
  • Abs to the thyroid microsomal antigen were traditionally based on partially purified antigen preparations used in complement-fixing assays, immunofluorescence tests or systems based on antigen-coated cells or particles (e.g. tanned red cell/haemagglutination techniques), antigen-coated enzyme linked immunosorbent assay (ELISA) plates or antigen-coated tubes.
  • ELISA enzyme linked immunosorbent assay
  • TPO had been identified as the major component of thyroid microsomal antigen
  • assays to measure TPO antibodies were more commonly based on purified preparations of native or recombinant TPO.
  • the currently used methods include ELISA and radioimmunoassay (RIA) techniques.
  • TPO autoantibodies are known to be markers of autoimmune thyroid disease (AITD) such as Graves' disease and Hashimoto's thyroiditis. Approx 80% of patients with AITD are found to have TPO Abs and the levels of TPO Abs in these patients are usually very high. However, TPO Abs, albeit at lower levels, are found in approx 20% of healthy female blood donors. The prevalence of TPO Abs increases with age, rising from approx 15% in the 18 to 24 age group, to approx 25% in the 55 to 64 age group for healthy females. The prevalence of TPO Abs in healthy male blood donors is approx 10%.
  • AITD autoimmune thyroid disease
  • Tg Abs from healthy blood donors and Tg Abs from patients with AITD appear to be directed to different epitopes on the Tg molecule.
  • autoantibody epitopes on human TPO as well as those on Tg are conformational
  • TPO Abs due to differences in reactivity of TPO Abs from healthy blood donors and TPO Abs from patients with AITD or other autoimmune diseases.
  • a method of monitoring the reactivity of TPO Abs which method comprises:
  • the modified TPO itself therefore contains corresponding truncations and/or deletions.
  • the TPO 90kD reference preparation and the modified TPO preparations are used.
  • the TPO preparations (reference and modified) can be labelled with different reagents (radiologically, for example with 1 5 I or 35 S methionine) or chemically, for example with chemiluminescent reagents, bioluminescent reagents, fluorescent compounds, enzymes, metal chelates or a dye (such as a streptavidin dye complex).
  • the difference in the ability of TPO Abs to react with reference TPO and modified TPO will indicate the presence of disease- related TPO Abs or disease-unrelated TPO Abs. It is thereby possible to distinguish between disease-related TPO Abs and disease-unrelated TPO Abs.
  • the autoantibodies are monitored using an ELISA.
  • ELISA diluted serum samples are added to TPO coated plastic wells of a standard ELISA plate and incubated to allow for TPO autoantibody binding to TPO on the well wall to occur.
  • Bound autoantibody is then quantified typically by addition of anti-human IgG (or protein A) coupled to an enzyme such as horseradish peroxidase or alkaline phosphatase, addition of an appropriate enzyme substrate which forms a coloured reaction product and quantification of the colour intensity using a spectrophotometer.
  • the colour intensity is a function of the amount of Abs in the serum sample.
  • a standard curve can be included in the assay to give fully quantitative results, or results can be expressed as an assay index in which the colour intensity (optical density or OD) in the test sample is divided by the OD observed for a standard reference TPO autoantibody preparation.
  • TPO Abs Another method which may be used for monitoring TPO Abs is a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • TPO Abs in a test serum bind to TPO coated tubes and are then detected by addition of either 125 I-labelled TPO, 125 I-labelled anti-human IgG or 125 I-labelled protein A.
  • sandwich-type systems in which TPO Abs in a test serum bind to TPO coated tubes and are then detected by addition of either 125 I-labelled TPO, 125 I-labelled anti-human IgG or 125 I-labelled protein A.
  • TPO monoclonal antibody can be coated onto the plastics tubes and the tubes will bind 125 I-labelled TPO.
  • TPO Abs in test sera will inhibit the binding of 125 I-TPO and results can be read off a standard curve.
  • TPO autoantibody assays depend on the direct liquid phase interaction between TPO Abs and 1 5 I-labelled antigen.
  • diluted serum samples are first incubated with 125 I-labelled TPO to allow formation of 125 I-labelled TPO/TPO autoantibody complexes in the liquid phase.
  • the labelled complexes are then precipitated by addition of solid phase protein A.
  • the amount of TPO Abs in the test serum is a function of the 125 I-labelled antigen precipitated and the results are read off a standard curve.
  • TPO labelled indirectly can also be used in assay systems to measure TPO Abs.
  • the modified TPO may be labelled with, for example, 35 S (for example, as 35 S-methionine), typically carried out using a methionine-free rabbit reticulocyte lysate system. Unreacted 35 S-methionine may be removed, for example, by passing through a Sephadex G50 column.
  • 35 S-methionine typically carried out using a methionine-free rabbit reticulocyte lysate system.
  • Unreacted 35 S-methionine may be removed, for example, by passing through a Sephadex G50 column.
  • other radioisotope labels may be used; as further alternatives enzyme labels or luminescent or fluorescent labels or dyes may be used.
  • the modified TPO genes are truncated and/or deleted by digestion at restriction enzyme sites and isolation of the resulting modified genes (typically on agarose gels).
  • the modified TPO genes containing truncations and/or deletions may be cloned into a pTZ18 vector under the control of a T7 promoter.
  • Other suitable vectors and/or promotors may be used if appropriate.
  • a Promega TnT transcription/translation kit may be used during the production and/or labelling of the modified TPO.
  • Other suitable protein expression systems may be used if appropriate.
  • Figure 1 shows an outline of restriction enzyme sites (frequency and position in the DNA sequence) used to introduce modifications into the TPO sequence
  • FIG. 2 shows the TPO DNA sequence/translated protein sequence (with selected restriction enzyme sites shown);
  • FIG. 3 is a schematic representation of modifications introduced into the TPO sequence
  • Figure 4a is a schematic map of an unmodified pTZ18 cloning vector suitable for use according to the invention.
  • Figure 4b is a pTZ18 vector DNA sequence and Linker sequence suitable for use according to the invention.
  • FIG. 5 shows a T7 promoter sequence suitable for use according to the invention.
  • Figure 6 is a summary of the reactivity of modified TPO with Abs in sera from patients with AITD, Addison's disease and healthy blood donors.
  • Sera from 20 patients with AITD (16 Graves' and 4 Hashimoto's) 8 patients with Addison's disease and 9 healthy blood donors were studied for reactivity with TPO Abs using an immunoprecipitation assay.
  • sera were incubated with the 35 S labelled variously modified TPO preparations for 2 hours at room temperature followed by incubation for 1 hour with solid phase protein A to bind 35 S modified TPO-TPO autoantibody complexes.
  • the protein A-bound complexes were then counted for 35 S and results computed as percentage binding of total radioactivity applied.
  • results are given as a percentage of binding of the 35 S-labelled 90kD TPO reference protein.
  • TPO gene Modifications to the TPO gene were carried out by restriction enzyme digestion at specific enzyme sites in the TPO sequence (see Figures 1 to 3). These truncated and/or deleted TPO gene sequences were then isolated and purified using a commercial GENECLEAN kit (GENECLEAN II, ANACHEM, Luton, Beds). Briefly, the digested TPO gene fragments were run on ethidium bromide stained agarose gels, and the relevant DNA bands excised from the gel using a scalpel blade and placed in a microfuge tube.
  • GENECLEAN kit GENECLEAN kit
  • the approximate volume of the gel slice was then estimated by weight and 3 volumes of 6M sodium iodide stock solution added.
  • the agarose slice was then dissolved by placing the microfuge tube at 55° C for 5 minutes.
  • lO ⁇ l of the kit GLASSMILK insoluble silica matrix stock
  • the GLASSMILK/DNA complex was then pelleted in a microfuge for 5 seconds. This pellet was then washed 3 times with GENECLEAN II NEW WASH solution before being eluted from the GLASSMILK matrix with sterile water at 55°C for 3 minutes.
  • the purified TPO gene fragments cloned into the modified pTZ18 vector ( Figures 4a and 4b), containing a unique linker, ( Figure 4b) under the control of the T7 promoter (see Figure 5) were then expressed and labelled with 35 S-methionine in an in vitro rabbit reticulocyte lystate system using a Promega TnT transcription/translation kit.
  • the reactions were carried out with a methionine-free rabbit reticulocyte lysate in the presence of 3S S-methionine (lOCi/L; Amersham).
  • the reaction mixture was then passed through a 0.5 x 16cm column of Sephadex G50 (Pharmacia), eluted with 150mmol/L Tris buffer pH8.3, containing (per litre) 200mmol of NaCl, lOmL of Tween-20 and lOg of bovine serum albumin (Tris buffer); lmL fractions were collected. The fractions collected were tested for reactivity with TPO Abs by immunoprecipitation assay (IP A), diluted to give about 30,000 counts/ min per 50/xL and stored in aliquots at - 70°C. Synthesis of proteins coded for by the various constructs was assessed by analysis on acrylamide gels (9%) in SDS followed by autoradiography.
  • the IPA was carried out with a 96-well filtration plate system incorporating 0.45 m (pore size) filters (Multiscreen, Millipore UK Limited). Aliquots of 50 ⁇ L of 35 S- TPO were incubated with aliquots of diluted serum (50 ⁇ L diluted 10 fold in Tris buffer) for 2 hours at room temperature with shaking. To each microtiter well was then added 50 ⁇ L of Protein A-Sepharose (Pharmacia), diluted 10 fold in Tris buffer and the samples were incubated for 1 hour more at room temperature with shaking. The plate was then placed on the suction unit (Millipore UK) and the immune complexes bound to Protein A-Sepharose were washed three times with 200 ⁇ L of Tris buffer.
  • Results were expressed as percent binding of total radioactivity added. In addition, results were expressed as percent binding to 90kDa TPO reference protein.
  • FIG. 3 is a schematic representation of modifications introduced into the TPO sequence.
  • 90kD reference TPO protein was produced by digestion at Accl site. C- terminal truncations were at Sad (AA772-838 deleted); at Clal (AA631-838 deleted); at S al (AA 515-838 deleted) and BamHI (AA455-838 deleted). Internal deletions were: Xmal/Sacl (AA 514-772 deleted); Apal/Apal (AA 386-652 deleted); Narl/Xmal (AA 166-517 deleted); Eco47i ⁇ /MluI (AA3-324 deleted) and Eco47III/NarI (AA 3-166 deleted).
  • Table 1 Summary of Modifications Introduced into the TPO Sequence
  • Table 2 shows results of TPO autoantibody binding to 90kD reference and modified TPO in AITD, Addison's disease and healthy blood donors. Results are expressed as % binding of total radioactivity added and are mean ⁇ SD of two separate experiments.
  • Table 3 shows TPO autoantibody binding to 90kD reference and modified TPO in AITD, Addison's disease and healthy blood donors. Results are expressed as % binding to 90kD TPO reference protein and are mean +SD of two separate experiments.
  • Figure 6 is a summary of the reactivity of modified TPO with Abs in sera from patients with AITD, Addison's disease and healthy blood donors. The results show that:
  • autoimmune diseases include insulin dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE), and myaesthenia gravis. These observations may allow differentiation between disease related and unrelated TPO Abs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de contrôle de la réactivité d'auto-anticorps (Abs) de la peroxydase thyroïdienne (TPO) dans le but de distinguer parmi des auto-anticorps de la peroxydase thyroïdienne, ceux associés à une maladie et ceux non associés à une maladie. Ledit procédé consiste a) à marquer une TPO modifiée, préparée au moyen de gènes de la TPO exprimés contenant des troncatures et/ou des délétions; et b) à contrôler la réactivité de la TPO modifiée et marquée en utilisant des auto-anticorps de la TPO présents dans un fluide biologique d'un patient.
PCT/GB1997/003014 1996-11-01 1997-11-03 Controle de la reactivite d'auto-anticorps de la peroxydase thyroidienne Ceased WO1998020354A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP52113998A JP2001503983A (ja) 1996-11-01 1997-11-03 甲状腺ペルオキシダーゼ自己抗体の反応性のモニタリング
EP97910562A EP0935756A2 (fr) 1996-11-01 1997-11-03 Controle de la reactivite d'auto-anticorps de la peroxydase thyroidienne

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9622772.3A GB9622772D0 (en) 1996-11-01 1996-11-01 Monitoring reactivity of thyroid peroxidase autoantibodies
GB9622772.3 1996-11-01

Publications (2)

Publication Number Publication Date
WO1998020354A2 true WO1998020354A2 (fr) 1998-05-14
WO1998020354A3 WO1998020354A3 (fr) 1999-02-11

Family

ID=10802294

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/003014 Ceased WO1998020354A2 (fr) 1996-11-01 1997-11-03 Controle de la reactivite d'auto-anticorps de la peroxydase thyroidienne

Country Status (4)

Country Link
EP (1) EP0935756A2 (fr)
JP (1) JP2001503983A (fr)
GB (1) GB9622772D0 (fr)
WO (1) WO1998020354A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000031535A3 (fr) * 1998-11-25 2000-09-08 Quest Diagnostics Invest Inc Compositions d'essai autoanticorps vis-a-vis de la thyroide peroxydase, procedes et kits
US7196181B1 (en) 1990-01-30 2007-03-27 Quest Diagnostics Investments, Inc. Sequences encoding novel human thyroid peroxidase proteins and polypeptides

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023073A1 (fr) * 1992-05-19 1993-11-25 The Regents Of The University Of Michigan Regions d'epitopes de thyroide-peroxydase

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7196181B1 (en) 1990-01-30 2007-03-27 Quest Diagnostics Investments, Inc. Sequences encoding novel human thyroid peroxidase proteins and polypeptides
WO2000031535A3 (fr) * 1998-11-25 2000-09-08 Quest Diagnostics Invest Inc Compositions d'essai autoanticorps vis-a-vis de la thyroide peroxydase, procedes et kits

Also Published As

Publication number Publication date
WO1998020354A3 (fr) 1999-02-11
GB9622772D0 (en) 1997-01-08
JP2001503983A (ja) 2001-03-27
EP0935756A2 (fr) 1999-08-18

Similar Documents

Publication Publication Date Title
JPH06501103A (ja) 生物学的液体中の抗体を決定する免疫学的分析方法とその方法を実施するキット
WO2010085763A1 (fr) Méthodes de détection d'anticorps associés à des maladies auto-immunes utilisant un antigène peptidique hétérogène en trois dimensions
He et al. Extracellular epitopes of platelet glycoprotein Ib alpha reactive with serum antibodies from patients with chronic idiopathic thrombocytopenic purpura
US5139932A (en) Method and a kit for the diagnosis of iga nephropathy
US6537760B1 (en) Receptor binding assay for detecting TSH-receptor auto-antibodies
US4929543A (en) Process for the determination of an antibody in human body fluids
EP0724726B1 (fr) Procede de determination d'un analyte et son utilisation pour la determination d'auto-anticorps anti-recepteurs de tsh dans le serum d'un patient
EP2402368A1 (fr) Peptides de citrulline histone et utilisations associées
US20100240076A1 (en) Immunoassay involving mutant antigens to reduce unspecific binding
CA2176007A1 (fr) Procede pour le dosage d'anticorps antiphospholipides et kit associe
CA2405448A1 (fr) Technique d'investigation du cancer par epreuve biologique d'un autoanticorps contre mdm2 et reactif
WO1998020354A2 (fr) Controle de la reactivite d'auto-anticorps de la peroxydase thyroidienne
EP1021721A1 (fr) Dosages des anticorps du recepteur de la tsh
Nakachi et al. Epitopes recognised by tissue transglutaminase antibodies in coeliac disease
Satoh et al. Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA‐dependent protein kinase catalytic subunit
Tack et al. Free and viral chromosome-bound simian virus 40 T antigen: changes in reactivity of specific antigenic determinants during lytic infection
Pérez et al. Synthesis of overlapping fibrin citrullinated peptides and their use for diagnosing rheumatoid arthritis
JP3510889B2 (ja) 糖尿病及び前糖尿症状の診断のための方法
AU666429B2 (en) Method and kit for the determination of antibodies
US20090317413A1 (en) Detection of human platelet antigen (hpa) antibodies
JPH0326787B2 (fr)
US5376533A (en) Methods and compositions for the detection of Addison's disease
NL8302708A (nl) Immunologische meetmethode en reagens.
Kurien et al. Autoantibody to the leucine zipper region of 52 kDa Ro/SSA binds native 60 kDa Ro/SSA: identification of a tertiary epitope with components from 60 kDa Ro/SSA and 52 kDa Ro/SSA
JPH08512407A (ja) ヒトの甲状腺パーオキシダーゼの定量的測定法及びキット

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

WWE Wipo information: entry into national phase

Ref document number: 09297530

Country of ref document: US

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1998 521139

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1997910562

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997910562

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997910562

Country of ref document: EP