WO2000025137A1 - Assays for autoantibodies - Google Patents

Assays for autoantibodies Download PDF

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Publication number
WO2000025137A1
WO2000025137A1 PCT/GB1999/003548 GB9903548W WO0025137A1 WO 2000025137 A1 WO2000025137 A1 WO 2000025137A1 GB 9903548 W GB9903548 W GB 9903548W WO 0025137 A1 WO0025137 A1 WO 0025137A1
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WO
WIPO (PCT)
Prior art keywords
antigen
autoantibody
antibody
immobilised
sample
Prior art date
Application number
PCT/GB1999/003548
Other languages
French (fr)
Inventor
Peter John Burne
Bernard Rees Smith
Original Assignee
Rsr Limited
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 Limited filed Critical Rsr Limited
Priority to JP2000578660A priority Critical patent/JP4426110B2/en
Priority to DE69930843T priority patent/DE69930843T2/en
Priority to EP99951000A priority patent/EP1071956B1/en
Priority to US09/582,524 priority patent/US7001775B1/en
Publication of WO2000025137A1 publication Critical patent/WO2000025137A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/971—Capture of complex after antigen-antibody reaction
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/973—Simultaneous determination of more than one analyte
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/815—Test for named compound or class of compounds
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/824—Immunological separation techniques

Definitions

  • the present invention is concerned with assays for screening a sample of body fluid for autoantibodies to various antigens.
  • the present invention is concerned with screening a sample of body fluid for autoantibodies associated with autoimmune thyroid disease.
  • tne autoantiDodies are directed to three different thyroid proteins, namely thyroid peroxidase (TPO) , thyroglobulm (Tg) and the receptor for thyroid stimulating hormone (TSHR) .
  • TPO thyroid peroxidase
  • Tg thyroglobulm
  • TSHR thyroid stimulating hormone
  • thyroid function is controlled by a feedback system involving the pituitary gland.
  • the pituitary secretes the hormone thyroid stimulating hormone (TSH) into the circulating bloo ⁇ .
  • TSK then acts on TSH receptors (TSHR) on the surfaces of thyroid cells m such a way as to stimulate the synthesis and release of thyroid hormones (which stimulate metabolic processes in almost all cells) .
  • TSH receptors TSH receptors
  • TSHR TSH receptors
  • This feedback mechanism allows circulating thyroid hormone levels to be maintained within close limits, thus ensuring good control over metabolic activity. In thyroid disease, however, the above feedback system is often distorted.
  • thyroid hormone levels are lower than normal and because these low levels do not suppress TSH release, circulating TSH levels are high.
  • thyroid over-activity hyperthyroidism
  • thyroid levels are higher than normal and these high levels cause TSH release to be suppressed more than normal and circulating TSH levels are low.
  • TPO and T Two different thyroid proteins
  • Screening for autoantibodies to TPO and/or autoantibodies to Tg is important m the diagnosis and management of the various forms of autoimmune hypothyroidism, including post-partum thyroiditis and the like.
  • This screening for TPO autoantibodies and/or Tg autoantibodies complements monitoring of circulating TSH levels or thyroid hormone levels which reflect the extent of thyroid under-activity and effectiveness of treatment.
  • Hyperthyroidism is also often caused by autoimmune attack on the thyroid but m this condition, autoantibodies are formed to the TSHR. These TSHR autoantibodies mimic the effects of TSH and cause circulating thyroid hormone levels to be high. Such high thyroid hormone levels act on the pituitary and suppress circulating TSH levels and consequently TSH levels are lower than normal. Screening for autoantibodies to the TSHR is important the diagnosis of autoimmune hyperthyroidism. As with autoimmune hypothyroidism, screening for TSHR autoantibodies complements monitoring of circulating TSH levels or thyroid hormone levels which reflect the extent of thyroid over-activity and effectiveness of treatment.
  • Tg levels are usually measured by assays which depend on monoclonal and/or polyclonal antibodies to Tg but if autoantibodies to Tg are present m patient test samples, these autoantibodies can interfere with the Tg assays, giving erroneous results. Consequently, screening for autoantibodies to Tg is often carried out at the same time as detection and monitoring of circulating Tg levels.
  • autoimmune diseases such as type 1 diabetes (where autoantibodies are formed to insulin, glutamic ac d decarboxylase and to the islet cell protein ICA512 or IA2) , celiac disease (where autoantibodies are formed to tissue transglutammase) , myasthenia gravis (where autoantibodies are formed to the acetylcholme receptor and to calcium channels) , systemic lupus erythematosus (where autoantibodies are formed to DNA and to various nuclear proteins), and the like.
  • type 1 diabetes where autoantibodies are formed to insulin, glutamic ac d decarboxylase and to the islet cell protein ICA512 or IA2
  • celiac disease where autoantibodies are formed to tissue transglutammase
  • myasthenia gravis where autoantibodies are formed to the acetylcholme receptor and to calcium channels
  • systemic lupus erythematosus where autoantibodies are formed to
  • radioactive labels m which the labelled antigen binds directly to the respective autoantibody
  • methods using radioactive labels m competition assays include methods using, for example, radioactive labels m which the labelled antigen binds directly to the respective autoantibody, or methods using radioactive labels m competition assays.
  • radioactive assays include those based on agglutination of particles coated with antigen.
  • sandwich type enzyme linked immunosorbent assays ELISA
  • sandwich type enzyme linked immunosorbent assays have used ELISA plates coated with antigen combination with an antl-human IgG reagent conjugated with an enzyme such as horseradish peroxidase.
  • the current assays can only be carried out away from the patient m specially equipped laboratories;
  • the current assays can only be carried out by experienced personnel and take several hours to complete .
  • a method of screening a sample of body fluid for at least one autoantibody to at least one antigen comprises:
  • step (a) providing a source of said at least one antigen to said autoantibody; (b) providing a substrate having immobilised thereto at least one antibody to said antigen of step (a) ;
  • step (c) contacting said antigen source of step (a) with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present said sample;
  • step (d) allowing said mixture obtained m step (c) to flow relative to said substrate of step (b) so as to allow said mixture to contact said antibody immobilised to said substrate;
  • step (e) providing labelling means so as to permit monitoring of binding of said autoantibody and said antigen present m said mixture obtained m step (c) ; and (f) monitoring said binding so as to provide an indication of the presence of said autoantibody said sample of body fluid.
  • a method according to the present invention is particularly suitable for use screening for at least one autoantibody associated with autoimmune thyroid disease and where the antigen comprises a thyroid protein.
  • the thyroid protein is selected from the group consisting of thyroid peroxidase (TPO) , thyroglobul (Tg) and thyroid stimulating hormone receptor (TSHR) , and even more advantageously the thyroid protein is selected from the group consisting of TPO and Tg .
  • a method of screening a sample of body fluid for at least one autoantibody to at least one antigen comprising a thyroid protein selected from the group consisting of TPO, Tg and TSHR which method comprises:
  • step (c) contacting said antigen source of step (a) with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present said sample;
  • step (d) allowing said mixture obtained step (c) to flow relative to said substrate of step (b) so as to allow said mixture to contact said antibody immobilised to said substrate;
  • step (e) providing labelling means so as to permit monitoring of binding of said autoantibody and said antigen present m said mixture obtained m step (c) ,- and (f) monitoring said binding so as to provide an indication of tne presence of said autoantibody said sample of body fluid.
  • a method according to the present invention preferably further comprises screening, m addition to the autoantibody screening, for at least one further biological marker present a sample of body fluid from a patient, which marker is indicative of an autoimmune (typically thyroid) disease.
  • Suitable biological markers can be selected from the group consisting of thyroid stimulating hormone (TSH), thyroxme, tri-iodothyronme , thyroglobulm
  • a method according to the present invention therefore, preferably further comprises screening for the presence of at least one of the group consisting of TSH, thyroxme, tri-iodothyronme, Tg and the like, m a sample of body fluid obtained from a patient being tested for an autoimmune thyroid disease.
  • Such screening for the presence of a further biological marker or markers can in the case of at least thyroxme and/or tri-iodothyronme, comprise screening for total or free (circulating) thyroxme and/or total or free
  • means for screening for the presence of such a further biological marker or markers may be provided remote from the substrate substantially as hereinbefore described and suitably such remote screening means can be provided by a separate kit .
  • a method according to the present invention is also suitable for use m screening for at least one autoantibody associated with non-thyroid autoimmune disease, such as autoantibodies associated with type 1 diabetes, celiac disease, myasthenia gravis, systemic lupus erythematosus and the like.
  • non-thyroid autoimmune disease such as autoantibodies associated with type 1 diabetes, celiac disease, myasthenia gravis, systemic lupus erythematosus and the like.
  • a method according to the present invention further employs at least one substantially non- immobilised antibody to the antigen and preferably a method according to the present invention comprises contacting step (c) the antigen source and the sample of body fluid with the at least one substantially non- immobilised antibody.
  • substantially non- lmmobilised antibody denotes an antibody that when provided m the mixture obtained step (c) can be allowed to flow relative to the substrate employed the present invention.
  • monitoring m step (f) of a method according to the present invention comprises observing a colorimet ⁇ c change dependent on the binding of the autoantibody and the antigen present m the mixture obtained by step (c) .
  • the labelling means can comprise a colorimetric label selected from the group consisting of colloidal gold, colloidal carbon, coloured latex, dyed polymers and the like.
  • the colorimetric label comprises colloidal gold.
  • the monitoring step (f) can involve electronic monitoring, whereby a visible readout can be oDtamed indicative of autoantibody and antigen binding.
  • the labelling means can further comprise a linker by which the colorimetric label, such as colloidal gold, may be attached to the antigen and/or antibody to be labelled.
  • a suitable linker can include -biotm-antibiotm- , -biotm- strepavidm- (SA) or the like.
  • SA -biotm-antibiotm-
  • SA -biotm- strepavidm-
  • the labelling means may be applied to an antigen and/or antibody to be labelled substantially remote from the substrate.
  • the labelling means may be provided to the substrate and such labelling means may be applied to the antigen and/or antibody to be labelled when the antigen and/or antibody has also been provided to the substrate.
  • Advantageously monitoring m step (f ) of a method according to the present invention can further comprise providing a positive control that is present m the presence or absence of the autoantibody or autoantibodies being screened.
  • the positive control may comprise, attaching to the substrate, a capture reagent for a colorimetric label, such as colloidal gold or the like.
  • the positive control may comprise attaching to the substrate at least one control antibody to the antigen, which control antibody binds to a site on the antigen distinct to a binding site thereof for the autoantibody or autoantibodies being screened.
  • the control antibody is preferably attached to the substrate employed m a method according to the present invention at a location of the substrate downstream relative to the immobilised antibody.
  • a further alternative positive control can comprise attaching to the substrate at least one control agent that can bind to the at least one substantially non-immobilised antibody.
  • the control agent can be selected from the group consisting of anti-mouse IgG, anti-human IgG or the like, and is typically attached to the substrate downstream relative to the immobilised antibody.
  • a method according to the present invention comprises allowing a mixture obtained m step (c) to flow along the substrate and interact with the antibody immobilised to the substrate. It is preferred that at least the sample of body fluid is contacted with an application zone of the substrate, which application zone is provided upstream on the substrate relative to the immobilised antibody, and wherein the mixture is allowed to flow from the application zone along the substrate so as to interact with the immobilised antibody.
  • the application zone can include the source of the antigen of step (a) , and the mixture in step (c) is obtained by contacting the sample of body fluid with the antigen of the application zone.
  • the application zone can further include the non- immobilised antibody, and the mixture in step (c) can be obtained by contacting said sample of body fluid and the antigen with the non-immobilised antibody present in the application zone.
  • the mixture of step (c) can be provided by contacting the antigen source of step (a) and the sample of body fluid substantially remote from the substrate and said mixture is subsequently contacted with the application zone of the substrate.
  • the antigen source of step (a) , the sample of body fluid and the non- immobilised antibody are contacted substantially remote from the substrate so as to provide the mixture of step (c) , and the mixture is subsequently contacted with the application zone of the substrate.
  • the sample of body fluid is screened for one autoantibody.
  • the antigen includes a binding site to which either the autoantibody or the immobilised antibody can bind, whereby in step (d) binding of the immobilised antibody to the binding site is substantially precluded where the autoantibody has substantially bound to the binding site in step (c) .
  • the sample of body fluid is screened for at least first and second autoantibodies to the antigen, wherein at least first and second antibodies to the antigen are immobilised to the substrate in step (b) .
  • the antigen includes:
  • step (d) binding of the first immobilised antibody to the first binding site is substantially precluded where the first autoantibody has substantially bound to the first binding site in step (c) ;
  • step (d) binding of the second immobilised antibody to the second binding site is substantially precluded where the second autoantibody has substantially bound to the second binding site in step (c) ;
  • first and second binding sites are substantially distinct sites on the antigen.
  • Labelling means can suitably be provided to the antigen m first or second screening methods substantially as described above.
  • the non- immobilised antibody can be provided with the labelling means, where the non- immobilised antibody is capable of binding to a site on the antigen substantially distinct from a binding site for either (i) the autoantibody or autoantibodies being screened or (ii) the immobilised antibody, whereby m step (d) , antigen is allowed to be substantially bound both to the immobilised antibody and to the non- immobilised antibody.
  • the non- immobilised antibody is capable of binding to a site on the antigen to which either the first or second autoantibody can bind and which is substantially distinct to a binding site on the antigen for the immobilised antibody, whereby m step (d) antigen is allowed to be substantially bound both to the immobilised antibody and to the non-immobilised antibody.
  • the antigen preferably includes: a first binding site to which either the first autoantibody or the immobilised antibody can bind, whereby m step (d) binding of immobilised antibody to the first binding site is substantially precluded where the first autoantibody has substantially bound to the first binding site m step (c) ; and
  • first and second binding sites are substantially distinct sites on the antigen.
  • the non- immobilised antibody is provided with the labelling means.
  • the immobilised antibody can comprise a first autoantibody to the antigen and the non-immobilised antibody can comprise a second autoantibody to the antigen.
  • the present invention further provides a kit for use m screening a sample of body fluid for at least one autoantibody to at least one antigen, which kit comprises:
  • kits according to the present invention are particularly suitable for use screening for at least one autoantibody associated with autoimmune (typically thyroid) disease and where the antigen comprises a thyroid protein.
  • the thyroid protein is selected from the group consisting of thyroid peroxidase (TPO) , thyroglobulm
  • thyroid stimulating hormone receptor Tg
  • Tg thyroid stimulating hormone receptor
  • TSHR thyroid stimulating hormone receptor
  • labelling means to permit monitoring of binding of the autoantibody and the antigen present m the mixture ; and (f) means for monitoring the binding so as to provide an indication of the presence of the autoantibody m the sample of body fluid.
  • kits according to the present invention further comprises means for screening for at least one further biological marker present m patient, which marKer is indicative of an autoimmune (typically thyroid) disease.
  • Suitable biological markers can be selected from the group consistmg of thyroid stimulating hormone (TSH) , thyroxme, tri-iodothyronme, thyroglobulm (Tg) and the like.
  • TSH thyroid stimulating hormone
  • Tg thyroglobulm
  • a kit according to the present invention further comprises means for screening for the presence of at least one of TSH, thyroxme, t ⁇ - lodothyronme, Tg and the like m the sample of body fluid.
  • kits according to the present invention further comprises a source of at least one substantially non- immobilised antibody to the antigen substantially as hereinbefore described and means whereby the non- lmmobilised antibody can be contacted with the antigen source and the sample of body fluid.
  • the monitoring means employed a kit according to the present invention comprise means for observing a colorimetric change dependent on the binding of the autoantibody and the antigen present said mixture substantially as hereinbefore described, although other monitoring means can be employed also substantially as hereinbefore described.
  • the labelling means can comprise a colorimetric label, such as colloidal gold substantially as hereinbefore described and a linker can be provided for attaching the colorimetric label to an antigen and/or antibody to be labelled again substantially as hereinbefore described.
  • the substrate may be provided with the labelling means for subsequent application.
  • the substrate of a kit according to the present invention can further comprise a positive control substantially as hereinbefore described with reference to a method according to the present invention.
  • the substrate of a kit according to the present invention can comprise an application zone provided upstream on the substrate relative to the immobilised antibody, whereby the mixture is allowed to flow from the application zone along the substrate so as to interact with the immobilised antibody.
  • that application zone can include the source of the antigen, and the mixture is obtained by contacting the sample of body fluid with the antigen of the application zone.
  • the application zone can further include the non-immobilised antibody, and means are provided whereby the mixture is obtained by contacting the sample of body fluid and the antigen with the non-immobilised antibody present in the application zone.
  • kits according to the present invention can comprise means whereby the antigen source and the sample of body fluid are contacted substantially remote from the substrate so as to provide the mixture and means whereby the mixture is subsequently contacted with the application zone.
  • a kit according to the present mvention comprises at least one substantially non- lmmobilised antibody
  • means can be provided whereby the antigen source, the sample of body fluid and / or the non- lmmobilised antibody are contacted substantially remote from the substrate so as to provide the mixture, and means whereby the mixture is subsequently contacted with the application zone.
  • a kit according to the present invention preferably further comprises wick means arranged downstream relative to the immobilised antibody so as to permit or potentiate flow of at least the sample of body fluid towards the immobilised antibody.
  • a first kit according to the present invention is suitable for screening for one autoantibody m the sample of body fluid and the antigen includes a binding site to which either the autoantibody or the immobilised antibody can bind, whereby binding of the immobilised antibody to the binding site is substantially precluded where the autoantibody has previously substantially bound to the binding site.
  • a second kit according to the present invention is suitable for screening the sample of body fluid for at least first and second autoantibodies to the antigen, wherein at least first and second antibodies to the antigen are immobilised to the substrate.
  • the antigen includes:
  • first and second binding sites are substantially distinct sites on the antigen.
  • the labelling means can be provided to the antigen of a first or second kit according to the present invention.
  • a first or second kit according to the present invention further comprises at least one substantially non-immobilised antibody
  • the non- lmmobilised antibody can be provided with the labelling means, which non- immobilised antibody is capable of binding to a site on the antigen substantially distinct from a binding site for either d) the autoantibody or autoantibodies being screened or (ii) the immobilised antibody, whereby antigen is allowed to be substantially bound both to the immobilised antibody and to the non- lmmobilised antibody.
  • a third kit according to the present invention is suitable for screening the sample of body fluid for at least first and second autoantibodies to the antigen, which third kit further comprises at least one substantially non- immobilised antibody, wherein the non-immobilised antibody is capable of binding to a site on the antigen to which either the first or second autoantibody can bind and which is substantially distinct to a binding site on the antigen for the immobilised antibody, whereby antigen is allowed to be substantially bound both to the immobilised antibody and to the non-immobilised antibody.
  • the antigen of the third kit includes :
  • first and second binding sites are substantially distinct sites on the antigen.
  • the non-immobilised antibody is provided with the labelling means in a third kit according to the present invention. It may be preferred that the immobilised antibody comprises a first autoantibody to the antigen and the non-immobilised antibody comprises a second autoantibody to the antigen.
  • Non-immobilised and immobilised antibodies employed in methods or kits according to the present invention are generally provided in substantially purified form and can comprise monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments, synthetic antibodies, substances mimicking antibodies , autoantibodies or the like.
  • Preferred aspects of the present invention comprise the non-immobilised antibody and/or the immobilised antibody comprising an autoantibody, which may preferably be a monoclonal antibody, and/or the non- immobilised antibody and/or the immobilised antibody can comprise a monoclonal antibody.
  • first and second screening methods, and first and second kits, according to the present invention in the presence of an autoantibody or autoantibodies being screened for in a sample of body fluid, generally binding of the autoantibody or autoantibodies with the antigen precludes binding of the latter with immobilised antibody.
  • a colorimetric label is employed in first and second screening methods and kits according to the present invention, substantially no colour change due to binding of antigen to immobilised antibody is thus seen in the presence of an autoantibody or autoantibodies; alternatively in the absence of an autoantibody or autoantibodies, a colour change is seen due to binding of antigen to immobilised antibody.
  • a colour change is seen indicative of antigen and immobilised antibody binding.
  • substantially no colour change can be seen, or some colour change can be seen due to non-immobilised antibody binding to antigen in competition with autoantibody and antigen binding whereby antigen bound to non-immobilised antibody can also bind to immobilised antibody giving a colour change .
  • an antigen employed in methods or kits of the present invention can comprise recombinant antigen, native antigen (autoantigen) , synthetic antigen, antigen fragments, substances mimicking antigen or the like.
  • a substrate for use in the present invention can comprise a membrane of nitrocellulose, cellulose acetate, a polyamide or the like.
  • the present invention comprises screening a sample of blood, plasma, serum or urine for at least one autoantibody .
  • the present invention further provides use of a kit substantially as hereinbefore described in screening a sample of body fluid for at least one autoantibody to at least one antigen again substantially as hereinbefore described.
  • a method of screening a patient for at least one autoantibody to at least one antigen comprises:
  • step (a) obtaining a sample of body fluid from the patient ;
  • step (b) contacting the sample of body fluid of step (a) with an antigen source of a kit substantially as hereinbefore described so as to obtain a mixture wherein said antigen is allowed to substantially bind with the autoantibody, when the latter is present in the sample;
  • step (c) allowing the mixture to flow relative to a substrate of the kit of step (b) so as to allow the mixture to contact the antibody immobilised to the substrate; and (d) monitoring binding of the autoantibody and the antigen present in the mixture, so as to provide an indication of the presence of the autoantibody in the sample of body fluid from the patient.
  • a method substantially as described above is preferably for testing the patient for an autoimmune thyroid disease and may preferably further comprise screening for the presence of at least one of TSH, thyroxine, tri-iodothyronine and Tg in the sample of body fluid obtained form the patient substantially as hereinbefore described.
  • a method of treating a patient suffering from, or susceptible to, an autoimmune disease which method comprises:
  • the autoimmune disease is a thyroid autoimmune disease and the therapeutically active substance comprises a pharmaceutical effective for treatment of thyroid autoimmune disease.
  • the mode of administration, dose and the like is generally at the discretion of an attendant physician.
  • m combination a kit substantially as hereinbefore described, and at least one therapeutically active substance effective for treatment of an autoimmune disease (typically a thyroid autoimmune disease) substantially as hereinbefore described.
  • an autoimmune disease typically a thyroid autoimmune disease
  • Figure la is a side view of a kit according to the present invention for screening TPO autoantibodies
  • Figure lb is a top view of the kit shown in Figure la;
  • Figures 2a and 2b are top views of the kit of Figures la and lb and respectively show the results obtained in the absence and presence of autoantibodies to TPO in the sample of body fluid;
  • Figures 3a and 3b are top views of a kit according to the present invention incorporating a positive control and show the results obtained in the absence and presence of autoantibodies to TPO;
  • Figure 4 is a top view of a kit according to the present invention for screening for Tg autoantibodies
  • Figure 5 is a top view of a kit according to the present invention for screening for both Tg autoantibodies and TPO autoantibodies ;
  • Figure 6 is a top view of a kit for screening for autoantibodies to different parts (epitopes) of TPO;
  • Figure 7a is a side view of a kit according to the present invention for screening for fist and second autoantibodies to Tg (autoantibodies Tg-AAbl and Tg-AAb2) ;
  • Figure 7b is a top view of the kit shown in Figure 7a;
  • Figures 8a, 8b, 8c and 8d are top views of the kit shown in Figures 7a and 7b and show the results obtained in the absence and presence of first and/or second autoantibodies to Tg (autoantibodies Tg-AAbl and Tg-AAb2) ;
  • Figure 9a is a side view of a kit according to the present invention for screening for first and second autoantibodies to TPO (autoantibodies TPO-AAbl and TPO-AAb2) ;
  • Figure 9b is a top view of the kit shown in Figure 9a.
  • Figures 10a and 10b are top views of the kit shown in Figures 9a and 9b and show the results obtained in the absence and presence of first and/or second autoantibodies to TPO (autoantibodies TPO-AAbl and TP0-AAb2) .
  • kits (1) for the screening for autoantibodies to TPO comprising a zone (2) for receiving a sample of body fluid (preferably blood) and includes a cell filter (not shown) for separating red blood cells from the remainder of the blood sample.
  • a pad (3) comprising strepavidin-gold (SA- gold) (4) is adjacent to receiving zone (2) .
  • Pad (3) is in communication with a zone comprising TPO-biotin (TPO-bi)
  • TPO (7) which is dried to a nitrocellulose membrane (6) .
  • purified antibodies to TPO (7) are immobilised to nitrocellulose membrane (6) and are located downstream from the zone comprising TPO-bi (5) .
  • a paper wick (8) is located at an opposite end to receiving zone (2) . Wick (8) is designed to permit or potentiate flow from receiving zone (2) towards purified immobilised antibodies to TPO (7) .
  • kit(l) shown in Figures la and lb in a screening method according to the present invention.
  • the first step comprises applying a sample of blood or the like to receiving zone (2) .
  • the sample of plasma will then flow towards wick (8) .
  • the blood cells are retained in receiving zone (2) .
  • the plasma then flows through pad (3) and forms a mixture with SA-gold (4) and this mixture flows towards TPO-bi (5) .
  • the plasma-SA-gold mixture arrives at the zone comprising TPO-bi (5) where TPO-bi (5) dissolves allowing formation of a TPO-bi-SA-gold complex. If autoantibodies to TPO are present in the plasma, these will bind to TPO in the TPO- bi-SA-gold complex.
  • Step 4 The mixture of plasma and TPO-bi-SA-gold complex then flows towards immobilised antibodies to TPO (7) .
  • Step 6 The reaction in the presence of autoantibodies to TPO allows autoantibodies to TPO in the plasma to bind to TPO-bi-SA-gold complex preventing the complex from binding to immobilised TPO antibodies (7) . Therefore, the absence of a red-gold line at the site of immobilised TPO antibodies (7) indicates the presence of TPO autoantibodies in the sample of body fluid.
  • FIG. 3a there can be seen an embodiment of the present invention which is extended to provide a positive control which is stained red irrespective of the presence or absence of TPO autoantibodies.
  • a rabbit antibody (11) to TPO is stained red-gold by a TPO-bi-SA- gold complex giving a red-gold line (12) . Therefore, as illustrated, in the absence of autoantibodies to TPO in the sample of body fluid to be tested, immobilised antibodies to TPO (7) are stained red-gold by the TPO-bi- SA-gold complex giving red-gold line (10) (which is also illustrated in Figure 2a) . Therefore, two red-gold lines (10, 12) will be indicative of a sample of body fluid which does not contain autoantibodies to TPO.
  • Figure 3b is identical to Figure 3a except that it illustrates a reaction where the sample of body fluid to be tested comprises autoantibodies to TPO.
  • autoantibodies to TPO in plasma bind to a TPO-bi-SA-gold complex thereby preventing the complex binding to immobilised antibodies to TPO (7) . Therefore, in this case, only one red-gold line (for the control) namely line (12), will be visible indicating the presence of TPO autoantibodies in the sample of body fluid.
  • Kit (13) is identical to kit (1) as shown in Figure 1, apart from the replacement of TPO-bi with Tg- bi and replacement of the immobilised antibodies to TPO with immobilised antibodies to Tg. More particularly, kit
  • Kit (13) similarly comprises receiving zone (2), pad (3) comprising SA-gold (4) and wick (8) .
  • Kit (13) further comprises a zone comprising Tg-bi (14) dried to nitrocellulose membrane (6) .
  • Purified antibodies to Tg (15) are immobilised to nitrocellulose membrane (6) and located downstream from Tg-bi (14) .
  • An immobilised rabbit antibody to Tg (16) is also present to provide a positive control .
  • kit (17) for screening for both autoantibodies to TPO and autoantibodies to Tg .
  • kit (17) comprises (as previously referred to in kits (1) and (13) receiving zone (2) , pad (3) comprising SA-gold (4) and wick (8) .
  • Kit (17) further comprises a zone including both TPO-bi (5) and Tg-bi (14) dried to nitrocellulose membrane (6) .
  • Purified antibodies to TPO (7) and purified antibodies to Tg (15) are immobilised to nitrocellulose membrane (6) and are located downstream from TPO-bi (5) and Tg-bi (14) .
  • Immobilised rabbit antibodies to TPO (11) and Tg (16) are also present to provide a positive control.
  • Figure 6 illustrates a kit (18) for screening for autoantibodies to different parts (first and second epitopes) of TPO.
  • This kit comprises immobilised antibodies to different autoantigenic epitopes on TPO which are used for the detection of autoantibodies to different parts (first and second epitopes) of TPO.
  • kit (18) comprises [as previously referred to m Kits (1), (13) and (17)] receiving zone (2), pad (3) comprises SA-gold (4) and wick (8) .
  • TPO-bi (5) is dried to nitrocellulose membrane (6) .
  • TPO antibodies to the first epitope (19) and TPO antibodies to the second epitope (20) are immobilised on nitrocellulose membrane (6) .
  • An immobilised rabbit antibody to TPO (11) is also present to provide a positive control .
  • kit (21) for screening for first and second autoantibodies to Tg (autoantibodies Tg-AAbl and Tg-AAb2) .
  • kit (21) comprises zone (2) for receiving a sample of body fluid, pad (3) , nitrocellulose membrane (6) and wick (8) .
  • Tg-gold denotes Tg previously labelled with -biotin- antibiotin-colloidal gold and subsequently applied to pad (3) .
  • Purified first and second antibodies (15a, 15b) are immobilised (immobilised antibodies Tg-Abl and Tg-Ab2) to nitrocellulose membrane (6) and are located downstream from Tg-gold (22) .
  • An immobilised rabbit antibody to Tg (16) is also present to provide a positive control.
  • Autoantibody Tg-AAbl binds to the same site of Tg-gold (22) as immobilised Tg-Abl (15a) .
  • Autoantibody Tg-AAb2 binds to the same site of Tg-gold (22) as immobilised Tg-Ab2 (15b) .
  • kit (21) shown in Figures 7a and 7b in a screening method according to the present invention.
  • the first step comprises applying a sample of blood or the like to receiving zone (2) .
  • the sample of plasma will then flow towards wick (8) .
  • the blood cells are retained in receiving zone ( 2 ) .
  • the plasma then f lows through pad ( 3 ) and forms a mixture with Tg-gold (22 ) and this mixture flows towards immobilised antibodies Tg-Abl and Tg-Ab2 ( 15a , 15b) .
  • the mixture of plasma and Tg-gold (22) reaches immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b) .
  • reaction m the absence of autoantibodies Tg-AAbl and Tg-AAb2 to Tg m the plasma allows Tg-gold (22) to bind to both immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b) , giving two red gold lines (23, 24) m addition to control line (25) for Tg rabbit antibody (16), as illustrated m Figure 8a.
  • reaction m the presence of autoantibody Tg-AAbl to Tg in the plasma allows autoantibody Tg-AAbl the plasma to bind to Tg-gold (22) preventing Tg-gold (22) from binding to immobilised antibody Tg-Abl (15a) .
  • No red gold line is seen at the site of immobilised antibody Tg-Abl and indicates the presence of autoantibody Tg-AAbl to Tg m the plasma.
  • This reaction m the absence of autoantibody Tg- AAb2 to Tg in the plasma allows Tg-gold (22) to bind to immobilised antibody Tg-Ab2 (15b) and a red gold line (24) is seen m addition to control line (25) , as illustrated m Figure 8b.
  • Step 7 The reaction in the presence of both autoantibodies Tg-AAbl and Tg-AAb2 to Tg in the plasma allows autoantibodies Tg- AAbl and Tg-AAb2 in the plasma to bind the Tg-gold (22) .
  • Tg-gold (22) is prevented from binding to immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b) .
  • No red gold lines are seen at the sites of immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b), indicating the presence of autoantibodies Tg-AAbl and Tg-AAb2 to Tg in the plasma.
  • Red gold control line (25) is seen, as illustrated in Figure 8d.
  • kit (26) comprises zone (2) for receiving a sample of body fluid, pad (3), nitrocellulose membrane (6) and wick
  • Non-immobilised antibody TPO-Abl-gold (27) denotes non-immobilised antibody TPO-Abl previously labelled with -biotin-antibiotin-colloidal gold and subsequently applied to pad (3) .
  • TPO (28) is dried to nitrocellulose membrane (6) .
  • Purified second antibody to TPO (29) is immobilised (immobilised antibody TPO-Ab2) to nitrocellulose membrane (6) and is located downstream of non-immobilised antibody TPO-Abl-gold (27) and TPO (28) .
  • Non-immobilised antibody TPO-Abl-gold binds to the same site of TPO (28) as autoantibody TPO-AAbl.
  • Immobilised antibody TPO-Ab2 binds to the same site of TPO (28) as autoantibody TPO-AAb2.
  • kit (26) shown in Figures 9a and 9b in a screening method according to the present invention.
  • the first step comprises applying a sample of blood or the like to receiving zone (2) .
  • the sample will then flow towards wick (8) .
  • the blood cells are retained in receiving zone (2) .
  • the plasma then flows through pad (3) and forms a mixture with non-immobilised antibody TPO-Abl-gold (27) and this mixture flows towards TPO (28) .
  • Step 3 The mixture of step 2 reaches TPO ( 28 ) and TPO ( 28 ) also dissolves the mixture.
  • Non- immobilised antibody TPO-Abl-gold (27) binds with TPO
  • the mixture of plasma, non- immobilised antibody TPO-Abl- gold (27) and TPO (28) reach immobilised antibody TPO-Ab2
  • TPO-AAb2 to TPO m the plasma allows TPO (28) to bind to non- immobilised antibody TPO-Abl-gold (27) and immobilised antibody TPO-Ab2 (29) .
  • a red gold line (30) at the location of immobilised antibody TPO-Ab2 (29) indicates the absence of autoantibodies TPO-AAbl and TPO-AAb2 m the plasma, as illustrated m Figure 10a.
  • TPO-AAbl and / or TPO-AAb2 prevent binding of TPO (28) with non-immobilised antibody TPO-Abl-gold (27) and / or immobilised antibody TPO-Ab2 (29) , no red gold line is seen as illustrated m Figure 10b.
  • This Example describes screening for autoantibodies to Tg using a kit as illustrated in Figures 7 and 8.
  • Tg autoantibodies can detect Tg autoantibodies in plasma from healthy blood donors, or sera from patients with systemic lupus erythematosus, at the same prevalence as the reference radioactive test.
  • the Example describes screening for autoantibodies to TPO using a kit as illustrated in Figures 9 and 10.
  • TPO autoantibodies m (a) healthy blood donors or

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Abstract

A method and a kit for screening a sample of body fluid for at least one autoantibody to at least one antigen. A source of at least one antigen to the autoantibody is provided. A substrate having immobilised thereto at least one antibody to the antigen is also provided. The antigen source is contacted with the sample of body fluid, so as to obtain a mixture wherein the antigen is allowed to substantially bind with the autoantibody, when the latter is present in the sample of body fluid. The mixture is allowed to flow relative to the substrate so as to allow the mixture to contact the antibody immobilised to the substrate. Labelling means are provided to permit monitoring of binding of the autoantibody and the antigen present in the mixture, so as to provide an indication of the presence of the autoantibody in the sample of body fluid.

Description

Assays for Autoantibodies
The present invention is concerned with assays for screening a sample of body fluid for autoantibodies to various antigens. In particular, the present invention is concerned with screening a sample of body fluid for autoantibodies associated with autoimmune thyroid disease.
Autoimmune diseases are characterised by the presence of circulating autoantibodies and in autoimmune thyroid disease, for example, tne autoantiDodies are directed to three different thyroid proteins, namely thyroid peroxidase (TPO) , thyroglobulm (Tg) and the receptor for thyroid stimulating hormone (TSHR) .
In tne absence of thyroid disease, thyroid function is controlled by a feedback system involving the pituitary gland. The pituitary secretes the hormone thyroid stimulating hormone (TSH) into the circulating blooα. TSK then acts on TSH receptors (TSHR) on the surfaces of thyroid cells m such a way as to stimulate the synthesis and release of thyroid hormones (which stimulate metabolic processes in almost all cells) . As circulating thyroid hormone levels rise these hormones act back on the pituitary to inhibit TSH release. This causes blood TSH levels to fall with the effect cf lowering blood thyroid hormone levels. This feedback mechanism allows circulating thyroid hormone levels to be maintained within close limits, thus ensuring good control over metabolic activity. In thyroid disease, however, the above feedback system is often distorted. For example, when the thyroid is under- active (hypothyroidism) , thyroid hormone levels are lower than normal and because these low levels do not suppress TSH release, circulating TSH levels are high. In the case of thyroid over-activity (hyperthyroidism) , thyroid levels are higher than normal and these high levels cause TSH release to be suppressed more than normal and circulating TSH levels are low.
Hypothyroidism is often caused by autoimmune attack on the thyroid and this attack is associated with the formation of autoantibodies to two different thyroid proteins (autoantigens) , namely TPO and T . Screening for autoantibodies to TPO and/or autoantibodies to Tg is important m the diagnosis and management of the various forms of autoimmune hypothyroidism, including post-partum thyroiditis and the like. This screening for TPO autoantibodies and/or Tg autoantibodies (which indicates the likely cause of thyroid underactivity) complements monitoring of circulating TSH levels or thyroid hormone levels which reflect the extent of thyroid under-activity and effectiveness of treatment.
Hyperthyroidism is also often caused by autoimmune attack on the thyroid but m this condition, autoantibodies are formed to the TSHR. These TSHR autoantibodies mimic the effects of TSH and cause circulating thyroid hormone levels to be high. Such high thyroid hormone levels act on the pituitary and suppress circulating TSH levels and consequently TSH levels are lower than normal. Screening for autoantibodies to the TSHR is important the diagnosis of autoimmune hyperthyroidism. As with autoimmune hypothyroidism, screening for TSHR autoantibodies complements monitoring of circulating TSH levels or thyroid hormone levels which reflect the extent of thyroid over-activity and effectiveness of treatment.
In patients with thyroid cancer, screening for circulating levels of Tg is often used as an indicator of the presence of any residual malignant thyroid tumour cells after treatment. Tg levels are usually measured by assays which depend on monoclonal and/or polyclonal antibodies to Tg but if autoantibodies to Tg are present m patient test samples, these autoantibodies can interfere with the Tg assays, giving erroneous results. Consequently, screening for autoantibodies to Tg is often carried out at the same time as detection and monitoring of circulating Tg levels.
Many examples of other (i.e. non thyroid) autoimmune diseases are known, such as type 1 diabetes (where autoantibodies are formed to insulin, glutamic ac d decarboxylase and to the islet cell protein ICA512 or IA2) , celiac disease (where autoantibodies are formed to tissue transglutammase) , myasthenia gravis (where autoantibodies are formed to the acetylcholme receptor and to calcium channels) , systemic lupus erythematosus (where autoantibodies are formed to DNA and to various nuclear proteins), and the like. Currently, several types of assay have been used to measure autoantibodies. These include methods using, for example, radioactive labels m which the labelled antigen binds directly to the respective autoantibody, or methods using radioactive labels m competition assays. Several non- radioactive assays have also been used, including those based on agglutination of particles coated with antigen. In addition, sandwich type enzyme linked immunosorbent assays (ELISA) are available. These sandwich type enzyme linked immunosorbent assays have used ELISA plates coated with antigen combination with an antl-human IgG reagent conjugated with an enzyme such as horseradish peroxidase.
However, there have been some major limitations associated with current assay methods for autoantibodies, for example : -
the current assays can only be carried out away from the patient m specially equipped laboratories; and
the current assays can only be carried out by experienced personnel and take several hours to complete .
It is therefore the aim of the present invention to provide an improved assay system which alleviates some of the aforementioned problems.
It is a further object of the present invention to provide simple and rapid assay methods for the monitoring of autoantibodies and also to provide diagnostic kits for use in the simple and rapid detection of autoantibodies for the diagnosis of autoimmune diseases. It is a further object of the present invention to provide an assay method that can be carried out near the point of patient care by personnel who do not have experience m laboratory procedures .
According to the present invention, therefore, there is provided a method of screening a sample of body fluid for at least one autoantibody to at least one antigen, which method comprises :
(a) providing a source of said at least one antigen to said autoantibody; (b) providing a substrate having immobilised thereto at least one antibody to said antigen of step (a) ;
(c) contacting said antigen source of step (a) with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present said sample;
(d) allowing said mixture obtained m step (c) to flow relative to said substrate of step (b) so as to allow said mixture to contact said antibody immobilised to said substrate;
(e) providing labelling means so as to permit monitoring of binding of said autoantibody and said antigen present m said mixture obtained m step (c) ; and (f) monitoring said binding so as to provide an indication of the presence of said autoantibody said sample of body fluid.
A method according to the present invention is particularly suitable for use screening for at least one autoantibody associated with autoimmune thyroid disease and where the antigen comprises a thyroid protein. Advantageously the thyroid protein is selected from the group consisting of thyroid peroxidase (TPO) , thyroglobul (Tg) and thyroid stimulating hormone receptor (TSHR) , and even more advantageously the thyroid protein is selected from the group consisting of TPO and Tg .
According to a particularly preferred aspect of the present invention there is provided a method of screening a sample of body fluid for at least one autoantibody to at least one antigen comprising a thyroid protein selected from the group consisting of TPO, Tg and TSHR, which method comprises:
(a) providing a source of said at least one antigen to said autoantibody;
(b) providing a substrate having immobilised thereto at least one antibody to said antigen of step
(a) ;
(c) contacting said antigen source of step (a) with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present said sample;
(d) allowing said mixture obtained step (c) to flow relative to said substrate of step (b) so as to allow said mixture to contact said antibody immobilised to said substrate;
(e) providing labelling means so as to permit monitoring of binding of said autoantibody and said antigen present m said mixture obtained m step (c) ,- and (f) monitoring said binding so as to provide an indication of tne presence of said autoantibody said sample of body fluid.
A method according to the present invention preferably further comprises screening, m addition to the autoantibody screening, for at least one further biological marker present a sample of body fluid from a patient, which marker is indicative of an autoimmune (typically thyroid) disease. Suitable biological markers can be selected from the group consisting of thyroid stimulating hormone (TSH), thyroxme, tri-iodothyronme , thyroglobulm
(Tg) and the like. A method according to the present invention, therefore, preferably further comprises screening for the presence of at least one of the group consisting of TSH, thyroxme, tri-iodothyronme, Tg and the like, m a sample of body fluid obtained from a patient being tested for an autoimmune thyroid disease.
Such screening for the presence of a further biological marker or markers, can in the case of at least thyroxme and/or tri-iodothyronme, comprise screening for total or free (circulating) thyroxme and/or total or free
(circulating) tri-iodothyronme . It may be desirable for means for screening for the presence of such a further biological marker or markers to be provided to the substrate employed m the present invention.
Alternatively, means for screening for the presence of such a further biological marker or markers may be provided remote from the substrate substantially as hereinbefore described and suitably such remote screening means can be provided by a separate kit .
A method according to the present invention is also suitable for use m screening for at least one autoantibody associated with non-thyroid autoimmune disease, such as autoantibodies associated with type 1 diabetes, celiac disease, myasthenia gravis, systemic lupus erythematosus and the like.
It is often preferred that a method according to the present invention further employs at least one substantially non- immobilised antibody to the antigen and preferably a method according to the present invention comprises contacting step (c) the antigen source and the sample of body fluid with the at least one substantially non- immobilised antibody. The term "substantially non- lmmobilised antibody" as used herein denotes an antibody that when provided m the mixture obtained step (c) can be allowed to flow relative to the substrate employed the present invention. Preferably monitoring m step (f) of a method according to the present invention comprises observing a colorimetπc change dependent on the binding of the autoantibody and the antigen present m the mixture obtained by step (c) . Suitably the labelling means can comprise a colorimetric label selected from the group consisting of colloidal gold, colloidal carbon, coloured latex, dyed polymers and the like. Preferably the colorimetric label comprises colloidal gold. Alternatively, the monitoring step (f) can involve electronic monitoring, whereby a visible readout can be oDtamed indicative of autoantibody and antigen binding.
In the case where a colorimetric label is employed substantially as described above, the labelling means can further comprise a linker by which the colorimetric label, such as colloidal gold, may be attached to the antigen and/or antibody to be labelled. For example, a suitable linker can include -biotm-antibiotm- , -biotm- strepavidm- (SA) or the like. The labelling means may be applied to an antigen and/or antibody to be labelled substantially remote from the substrate. Alternatively, the labelling means may be provided to the substrate and such labelling means may be applied to the antigen and/or antibody to be labelled when the antigen and/or antibody has also been provided to the substrate.
Advantageously monitoring m step (f ) of a method according to the present invention can further comprise providing a positive control that is present m the presence or absence of the autoantibody or autoantibodies being screened.
For example, the positive control may comprise, attaching to the substrate, a capture reagent for a colorimetric label, such as colloidal gold or the like. Alternatively, the positive control may comprise attaching to the substrate at least one control antibody to the antigen, which control antibody binds to a site on the antigen distinct to a binding site thereof for the autoantibody or autoantibodies being screened. The control antibody is preferably attached to the substrate employed m a method according to the present invention at a location of the substrate downstream relative to the immobilised antibody. A further alternative positive control can comprise attaching to the substrate at least one control agent that can bind to the at least one substantially non-immobilised antibody. Suitably the control agent can be selected from the group consisting of anti-mouse IgG, anti-human IgG or the like, and is typically attached to the substrate downstream relative to the immobilised antibody.
Suitably, a method according to the present invention comprises allowing a mixture obtained m step (c) to flow along the substrate and interact with the antibody immobilised to the substrate. It is preferred that at least the sample of body fluid is contacted with an application zone of the substrate, which application zone is provided upstream on the substrate relative to the immobilised antibody, and wherein the mixture is allowed to flow from the application zone along the substrate so as to interact with the immobilised antibody.
Aptly, the application zone can include the source of the antigen of step (a) , and the mixture in step (c) is obtained by contacting the sample of body fluid with the antigen of the application zone. In the case where at least one substantially non- immobilised antibody is employed in a method according to the present invention, the application zone can further include the non- immobilised antibody, and the mixture in step (c) can be obtained by contacting said sample of body fluid and the antigen with the non-immobilised antibody present in the application zone.
Alternatively, the mixture of step (c) can be provided by contacting the antigen source of step (a) and the sample of body fluid substantially remote from the substrate and said mixture is subsequently contacted with the application zone of the substrate. In the case where at least one substantially non- immobilised antibody is employed in a method according to the present invention, the antigen source of step (a) , the sample of body fluid and the non- immobilised antibody are contacted substantially remote from the substrate so as to provide the mixture of step (c) , and the mixture is subsequently contacted with the application zone of the substrate.
In a first screening method according to the present invention, the sample of body fluid is screened for one autoantibody. Preferably the antigen includes a binding site to which either the autoantibody or the immobilised antibody can bind, whereby in step (d) binding of the immobilised antibody to the binding site is substantially precluded where the autoantibody has substantially bound to the binding site in step (c) .
In a second screening method according to the present invention, the sample of body fluid is screened for at least first and second autoantibodies to the antigen, wherein at least first and second antibodies to the antigen are immobilised to the substrate in step (b) . Preferably, the antigen includes:
a first binding site to which either the first autoantibody or the first immobilised antibody can bind, whereby in step (d) binding of the first immobilised antibody to the first binding site is substantially precluded where the first autoantibody has substantially bound to the first binding site in step (c) ; and
a second binding site to which either the second autoantibody or the second immobilised antibody can bind, whereby in step (d) binding of the second immobilised antibody to the second binding site is substantially precluded where the second autoantibody has substantially bound to the second binding site in step (c) ;
wherein the first and second binding sites are substantially distinct sites on the antigen.
Labelling means can suitably be provided to the antigen m first or second screening methods substantially as described above. Alternatively, where at least one substantially non- immobilised antibody is employed first or second screening methods according to the present invention, the non- immobilised antibody can be provided with the labelling means, where the non- immobilised antibody is capable of binding to a site on the antigen substantially distinct from a binding site for either (i) the autoantibody or autoantibodies being screened or (ii) the immobilised antibody, whereby m step (d) , antigen is allowed to be substantially bound both to the immobilised antibody and to the non- immobilised antibody.
In a third screening method according to the present invention for screening the sample of body fluid for at least first and second autoantibodies to the antigen, and where at least one substantially non-immobilised antibody is employed, the non- immobilised antibody is capable of binding to a site on the antigen to which either the first or second autoantibody can bind and which is substantially distinct to a binding site on the antigen for the immobilised antibody, whereby m step (d) antigen is allowed to be substantially bound both to the immobilised antibody and to the non-immobilised antibody.
In the third screening method according to the present invention, the antigen preferably includes: a first binding site to which either the first autoantibody or the immobilised antibody can bind, whereby m step (d) binding of immobilised antibody to the first binding site is substantially precluded where the first autoantibody has substantially bound to the first binding site m step (c) ; and
a second binding site to which either the second autoantibody or the non- immobilised antibody can bind;
wherein the first and second binding sites are substantially distinct sites on the antigen.
In the third screening method according to the present invention, preferably the non- immobilised antibody is provided with the labelling means. Suitably the immobilised antibody can comprise a first autoantibody to the antigen and the non-immobilised antibody can comprise a second autoantibody to the antigen.
The present invention further provides a kit for use m screening a sample of body fluid for at least one autoantibody to at least one antigen, which kit comprises:
(a) a source of the at least one antigen to the autoantibody;
(b) a substrate having immobilised thereto at least one antibody to the antigen;
(c) means for contacting the antigen source with the sample of body fluid, so as to obtain a mixture wherem the antigen is allowed to substantially bind with the autoantibody, when the latter is present m the sample;
(d) means for allowing the mixture to flow relative to the substrate so as to allow the mixture to contact the antibody immobilised to said substrate;
(e) labelling means to permit monitoring of binding of the autoantibody and the antigen present m the mixture; and
(f) means for monitoring the binding so as to provide an indication of the presence of the autoantibody m the sample of body fluid.
Substantially as hereinbefore described with reference to a method of screening according to the present invention, a kit according to the present invention is particularly suitable for use screening for at least one autoantibody associated with autoimmune (typically thyroid) disease and where the antigen comprises a thyroid protein.
Advantageously the thyroid protein is selected from the group consisting of thyroid peroxidase (TPO) , thyroglobulm
(Tg) and thyroid stimulating hormone receptor (TSHR) , and even more advantageously the thyroid protein is selected from the group consisting of TPO and Tg .
According to a preferred embodiment of the present invention there is provided a kit for use m screening a sample of body fluid for at least one autoantibody to at least one antigen comprising a thyroid protein selected fro the group consisting of TPO, Tg and TSHR, which kit comprises :
(a) a source of the at least one antigen to the autoantibody;
(b) a substrate having immobilised thereto at least one antibody to the antigen;
(c) means for contacting the antigen source with the sample of body fluid, so as to obtain a mixture wherein the antigen is allowed to substantially bind with the autoantibody, when the latter is present m the sample;
(d) means for allowing the mixture to flow relative to the substrate so as to allow the mixture to contact the antibody immobilised to the substrate;
(e) labelling means to permit monitoring of binding of the autoantibody and the antigen present m the mixture ; and (f) means for monitoring the binding so as to provide an indication of the presence of the autoantibody m the sample of body fluid.
Substantially as hereinbefore described with reference to a method of screening according to the present invention a kit according to the present invention further comprises means for screening for at least one further biological marker present m patient, which marKer is indicative of an autoimmune (typically thyroid) disease. Suitable biological markers can be selected from the group consistmg of thyroid stimulating hormone (TSH) , thyroxme, tri-iodothyronme, thyroglobulm (Tg) and the like. Preferably, therefore, a kit according to the present invention further comprises means for screening for the presence of at least one of TSH, thyroxme, tπ- lodothyronme, Tg and the like m the sample of body fluid.
Suitably, a kit according to the present invention further comprises a source of at least one substantially non- immobilised antibody to the antigen substantially as hereinbefore described and means whereby the non- lmmobilised antibody can be contacted with the antigen source and the sample of body fluid.
Advantageously, the monitoring means employed a kit according to the present invention comprise means for observing a colorimetric change dependent on the binding of the autoantibody and the antigen present said mixture substantially as hereinbefore described, although other monitoring means can be employed also substantially as hereinbefore described. Suitably the labelling means can comprise a colorimetric label, such as colloidal gold substantially as hereinbefore described and a linker can be provided for attaching the colorimetric label to an antigen and/or antibody to be labelled again substantially as hereinbefore described. In the case where labelling means are applied to an antigen and/or antibody following application thereof to the substrate, the substrate may be provided with the labelling means for subsequent application. Preferably the substrate of a kit according to the present invention can further comprise a positive control substantially as hereinbefore described with reference to a method according to the present invention.
Preferably the substrate of a kit according to the present invention can comprise an application zone provided upstream on the substrate relative to the immobilised antibody, whereby the mixture is allowed to flow from the application zone along the substrate so as to interact with the immobilised antibody.
Suitably, that application zone can include the source of the antigen, and the mixture is obtained by contacting the sample of body fluid with the antigen of the application zone. In the case where a kit according to the present invention further comprises at least one substantially non- immobilised antibody substantially as hereinbefore described, the application zone can further include the non-immobilised antibody, and means are provided whereby the mixture is obtained by contacting the sample of body fluid and the antigen with the non-immobilised antibody present in the application zone.
Alternatively, a kit according to the present invention can comprise means whereby the antigen source and the sample of body fluid are contacted substantially remote from the substrate so as to provide the mixture and means whereby the mixture is subsequently contacted with the application zone. In the case where a kit according to the present mvention comprises at least one substantially non- lmmobilised antibody, means can be provided whereby the antigen source, the sample of body fluid and / or the non- lmmobilised antibody are contacted substantially remote from the substrate so as to provide the mixture, and means whereby the mixture is subsequently contacted with the application zone.
A kit according to the present invention preferably further comprises wick means arranged downstream relative to the immobilised antibody so as to permit or potentiate flow of at least the sample of body fluid towards the immobilised antibody.
A first kit according to the present invention is suitable for screening for one autoantibody m the sample of body fluid and the antigen includes a binding site to which either the autoantibody or the immobilised antibody can bind, whereby binding of the immobilised antibody to the binding site is substantially precluded where the autoantibody has previously substantially bound to the binding site.
A second kit according to the present invention is suitable for screening the sample of body fluid for at least first and second autoantibodies to the antigen, wherein at least first and second antibodies to the antigen are immobilised to the substrate. Preferably the antigen includes:
a first binding site to which either the first autoantibody or the first immobilised antibody can bind, whereby binding of the first immobilised antibody to the first binding site is substantially precluded where the first autoantibody has previously substantially bound to the first binding site; and
a second binding site to which either the second autoantibody or the second immobilised antibody can bind, whereby binding of the second immobilised antibody to the second binding site is substantially precluded where the second autoantibody has previously substantially bound to the second binding site;
wherein the first and second binding sites are substantially distinct sites on the antigen.
Suitably the labelling means can be provided to the antigen of a first or second kit according to the present invention. Alternatively, where a first or second kit according to the present invention further comprises at least one substantially non-immobilised antibody, the non- lmmobilised antibody can be provided with the labelling means, which non- immobilised antibody is capable of binding to a site on the antigen substantially distinct from a binding site for either d) the autoantibody or autoantibodies being screened or (ii) the immobilised antibody, whereby antigen is allowed to be substantially bound both to the immobilised antibody and to the non- lmmobilised antibody. A third kit according to the present invention is suitable for screening the sample of body fluid for at least first and second autoantibodies to the antigen, which third kit further comprises at least one substantially non- immobilised antibody, wherein the non-immobilised antibody is capable of binding to a site on the antigen to which either the first or second autoantibody can bind and which is substantially distinct to a binding site on the antigen for the immobilised antibody, whereby antigen is allowed to be substantially bound both to the immobilised antibody and to the non-immobilised antibody.
Preferably the antigen of the third kit includes :
a first binding site to which either the first autoantibody or the immobilised antibody can bind, whereby binding of immobilised antibody to the first binding site is substantially precluded where the first autoantibody has previously substantially bound to the first binding site; and
a second binding site to which either the second autoantibody or the non-immobilised antibody can bind;
wherein the first and second binding sites are substantially distinct sites on the antigen.
Suitably the non-immobilised antibody is provided with the labelling means in a third kit according to the present invention. It may be preferred that the immobilised antibody comprises a first autoantibody to the antigen and the non-immobilised antibody comprises a second autoantibody to the antigen.
Non-immobilised and immobilised antibodies employed in methods or kits according to the present invention are generally provided in substantially purified form and can comprise monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments, synthetic antibodies, substances mimicking antibodies , autoantibodies or the like. Preferred aspects of the present invention comprise the non-immobilised antibody and/or the immobilised antibody comprising an autoantibody, which may preferably be a monoclonal antibody, and/or the non- immobilised antibody and/or the immobilised antibody can comprise a monoclonal antibody.
In first and second screening methods, and first and second kits, according to the present invention , in the presence of an autoantibody or autoantibodies being screened for in a sample of body fluid, generally binding of the autoantibody or autoantibodies with the antigen precludes binding of the latter with immobilised antibody. In the case where a colorimetric label is employed in first and second screening methods and kits according to the present invention, substantially no colour change due to binding of antigen to immobilised antibody is thus seen in the presence of an autoantibody or autoantibodies; alternatively in the absence of an autoantibody or autoantibodies, a colour change is seen due to binding of antigen to immobilised antibody.
In a third screening method and kit according to the present invention, again in the absence of autoantibody or autoantibodies being screened, a colour change is seen indicative of antigen and immobilised antibody binding. In the presence of autoantibody or autoantibodies, substantially no colour change can be seen, or some colour change can be seen due to non-immobilised antibody binding to antigen in competition with autoantibody and antigen binding whereby antigen bound to non-immobilised antibody can also bind to immobilised antibody giving a colour change .
Suitably an antigen employed in methods or kits of the present invention can comprise recombinant antigen, native antigen (autoantigen) , synthetic antigen, antigen fragments, substances mimicking antigen or the like.
A substrate for use in the present invention can comprise a membrane of nitrocellulose, cellulose acetate, a polyamide or the like.
Generally the present invention comprises screening a sample of blood, plasma, serum or urine for at least one autoantibody .
The present invention further provides use of a kit substantially as hereinbefore described in screening a sample of body fluid for at least one autoantibody to at least one antigen again substantially as hereinbefore described.
There is still further provided by the present invention a method of screening a patient for at least one autoantibody to at least one antigen, which method comprises:
(a) obtaining a sample of body fluid from the patient ; (b) contacting the sample of body fluid of step (a) with an antigen source of a kit substantially as hereinbefore described so as to obtain a mixture wherein said antigen is allowed to substantially bind with the autoantibody, when the latter is present in the sample;
(c) allowing the mixture to flow relative to a substrate of the kit of step (b) so as to allow the mixture to contact the antibody immobilised to the substrate; and (d) monitoring binding of the autoantibody and the antigen present in the mixture, so as to provide an indication of the presence of the autoantibody in the sample of body fluid from the patient.
A method substantially as described above is preferably for testing the patient for an autoimmune thyroid disease and may preferably further comprise screening for the presence of at least one of TSH, thyroxine, tri-iodothyronine and Tg in the sample of body fluid obtained form the patient substantially as hereinbefore described. There is further provided by the present invention a method of treating a patient suffering from, or susceptible to, an autoimmune disease, which method comprises:
screening the patient for at least one autoantibody to at least one antigen substantially as hereinbefore described; and
when at least one autoantibody is detected m a sample of body fluid obtained from the patient at a level indicative of an autoimmune disease, administering to the patient at least one therapeutically active substance effective m the treatment of the autoimmune disease .
Substantially as hereinbefore described the autoimmune disease is a thyroid autoimmune disease and the therapeutically active substance comprises a pharmaceutical effective for treatment of thyroid autoimmune disease. The mode of administration, dose and the like is generally at the discretion of an attendant physician.
There is still further provided by the present invention, m combination, a kit substantially as hereinbefore described, and at least one therapeutically active substance effective for treatment of an autoimmune disease (typically a thyroid autoimmune disease) substantially as hereinbefore described.
The present invention will now be illustrated with reference to the accompanying figures, which are given by way of example only.
Figure la is a side view of a kit according to the present invention for screening TPO autoantibodies;
Figure lb is a top view of the kit shown in Figure la;
Figures 2a and 2b are top views of the kit of Figures la and lb and respectively show the results obtained in the absence and presence of autoantibodies to TPO in the sample of body fluid;
Figures 3a and 3b are top views of a kit according to the present invention incorporating a positive control and show the results obtained in the absence and presence of autoantibodies to TPO;
Figure 4 is a top view of a kit according to the present invention for screening for Tg autoantibodies;
Figure 5 is a top view of a kit according to the present invention for screening for both Tg autoantibodies and TPO autoantibodies ;
Figure 6 is a top view of a kit for screening for autoantibodies to different parts (epitopes) of TPO;
Figure 7a is a side view of a kit according to the present invention for screening for fist and second autoantibodies to Tg (autoantibodies Tg-AAbl and Tg-AAb2) ;
Figure 7b is a top view of the kit shown in Figure 7a;
Figures 8a, 8b, 8c and 8d are top views of the kit shown in Figures 7a and 7b and show the results obtained in the absence and presence of first and/or second autoantibodies to Tg (autoantibodies Tg-AAbl and Tg-AAb2) ;
Figure 9a is a side view of a kit according to the present invention for screening for first and second autoantibodies to TPO (autoantibodies TPO-AAbl and TPO-AAb2) ;
Figure 9b is a top view of the kit shown in Figure 9a; and
Figures 10a and 10b are top views of the kit shown in Figures 9a and 9b and show the results obtained in the absence and presence of first and/or second autoantibodies to TPO (autoantibodies TPO-AAbl and TP0-AAb2) .
Referring firstly to Figures la and lb, there is shown a kit (1) for the screening for autoantibodies to TPO comprising a zone (2) for receiving a sample of body fluid (preferably blood) and includes a cell filter (not shown) for separating red blood cells from the remainder of the blood sample. A pad (3) comprising strepavidin-gold (SA- gold) (4) is adjacent to receiving zone (2) . Pad (3) is in communication with a zone comprising TPO-biotin (TPO-bi)
(5) which is dried to a nitrocellulose membrane (6) . Furthermore, purified antibodies to TPO (7) are immobilised to nitrocellulose membrane (6) and are located downstream from the zone comprising TPO-bi (5) . A paper wick (8) is located at an opposite end to receiving zone (2) . Wick (8) is designed to permit or potentiate flow from receiving zone (2) towards purified immobilised antibodies to TPO (7) .
The following series of steps illustrate the use of kit(l) shown in Figures la and lb in a screening method according to the present invention.
Step 1
The first step comprises applying a sample of blood or the like to receiving zone (2) . The sample of plasma will then flow towards wick (8) .
Step 2
The blood cells are retained in receiving zone (2) . The plasma then flows through pad (3) and forms a mixture with SA-gold (4) and this mixture flows towards TPO-bi (5) .
Step 3
The plasma-SA-gold mixture arrives at the zone comprising TPO-bi (5) where TPO-bi (5) dissolves allowing formation of a TPO-bi-SA-gold complex. If autoantibodies to TPO are present in the plasma, these will bind to TPO in the TPO- bi-SA-gold complex.
Step 4 The mixture of plasma and TPO-bi-SA-gold complex then flows towards immobilised antibodies to TPO (7) .
Step 5
The reaction in the absence of autoantibodies to TPO allows the TPO-bi-SA-gold complex to bind to immobilised antibodies to TPO (7) giving a red-gold line (10) , as illustrated in Figure 2a.
Step 6 The reaction in the presence of autoantibodies to TPO allows autoantibodies to TPO in the plasma to bind to TPO-bi-SA-gold complex preventing the complex from binding to immobilised TPO antibodies (7) . Therefore, the absence of a red-gold line at the site of immobilised TPO antibodies (7) indicates the presence of TPO autoantibodies in the sample of body fluid.
Referring now to Figure 3a, there can be seen an embodiment of the present invention which is extended to provide a positive control which is stained red irrespective of the presence or absence of TPO autoantibodies. A rabbit antibody (11) to TPO is stained red-gold by a TPO-bi-SA- gold complex giving a red-gold line (12) . Therefore, as illustrated, in the absence of autoantibodies to TPO in the sample of body fluid to be tested, immobilised antibodies to TPO (7) are stained red-gold by the TPO-bi- SA-gold complex giving red-gold line (10) (which is also illustrated in Figure 2a) . Therefore, two red-gold lines (10, 12) will be indicative of a sample of body fluid which does not contain autoantibodies to TPO. Figure 3b is identical to Figure 3a except that it illustrates a reaction where the sample of body fluid to be tested comprises autoantibodies to TPO. In this situation, autoantibodies to TPO in plasma bind to a TPO-bi-SA-gold complex thereby preventing the complex binding to immobilised antibodies to TPO (7) . Therefore, in this case, only one red-gold line (for the control) namely line (12), will be visible indicating the presence of TPO autoantibodies in the sample of body fluid.
Figure 4 illustrates a kit (13) for screening for Tg autoantibodies. Kit (13) is identical to kit (1) as shown in Figure 1, apart from the replacement of TPO-bi with Tg- bi and replacement of the immobilised antibodies to TPO with immobilised antibodies to Tg. More particularly, kit
(13) similarly comprises receiving zone (2), pad (3) comprising SA-gold (4) and wick (8) . Kit (13) further comprises a zone comprising Tg-bi (14) dried to nitrocellulose membrane (6) . Purified antibodies to Tg (15) are immobilised to nitrocellulose membrane (6) and located downstream from Tg-bi (14) . An immobilised rabbit antibody to Tg (16) is also present to provide a positive control .
Figure 5 illustrates a single kit (17) for screening for both autoantibodies to TPO and autoantibodies to Tg . More particularly, kit (17) comprises (as previously referred to in kits (1) and (13) receiving zone (2) , pad (3) comprising SA-gold (4) and wick (8) . Kit (17) further comprises a zone including both TPO-bi (5) and Tg-bi (14) dried to nitrocellulose membrane (6) . Purified antibodies to TPO (7) and purified antibodies to Tg (15) are immobilised to nitrocellulose membrane (6) and are located downstream from TPO-bi (5) and Tg-bi (14) . Immobilised rabbit antibodies to TPO (11) and Tg (16) are also present to provide a positive control.
Figure 6 illustrates a kit (18) for screening for autoantibodies to different parts (first and second epitopes) of TPO. This kit comprises immobilised antibodies to different autoantigenic epitopes on TPO which are used for the detection of autoantibodies to different parts (first and second epitopes) of TPO. More particularly, kit (18) comprises [as previously referred to m Kits (1), (13) and (17)] receiving zone (2), pad (3) comprises SA-gold (4) and wick (8) . TPO-bi (5) is dried to nitrocellulose membrane (6) .
TPO antibodies to the first epitope (19) and TPO antibodies to the second epitope (20) are immobilised on nitrocellulose membrane (6) . An immobilised rabbit antibody to TPO (11) is also present to provide a positive control .
Referring to Figures 7a and 7b, there is shown a kit (21) for screening for first and second autoantibodies to Tg (autoantibodies Tg-AAbl and Tg-AAb2) . As previously referred to m kits (1) , (13) , (17) and (18) , kit (21) comprises zone (2) for receiving a sample of body fluid, pad (3) , nitrocellulose membrane (6) and wick (8) . Tg-gold
(22) is present on pad (3) and is provided adjacent to zone
(2) . Tg-gold denotes Tg previously labelled with -biotin- antibiotin-colloidal gold and subsequently applied to pad (3) .
Purified first and second antibodies (15a, 15b) are immobilised (immobilised antibodies Tg-Abl and Tg-Ab2) to nitrocellulose membrane (6) and are located downstream from Tg-gold (22) . An immobilised rabbit antibody to Tg (16) is also present to provide a positive control.
Autoantibody Tg-AAbl binds to the same site of Tg-gold (22) as immobilised Tg-Abl (15a) . Autoantibody Tg-AAb2 binds to the same site of Tg-gold (22) as immobilised Tg-Ab2 (15b) .
The following series of steps illustrate the use of kit (21) shown in Figures 7a and 7b in a screening method according to the present invention.
Step 1
The first step comprises applying a sample of blood or the like to receiving zone (2) . The sample of plasma will then flow towards wick (8) .
Step 2
The blood cells are retained in receiving zone ( 2 ) . The plasma then f lows through pad ( 3 ) and forms a mixture with Tg-gold (22 ) and this mixture flows towards immobilised antibodies Tg-Abl and Tg-Ab2 ( 15a , 15b) . Step 3
The mixture of plasma and Tg-gold (22) reaches immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b) .
Step 4
The reaction m the absence of autoantibodies Tg-AAbl and Tg-AAb2 to Tg m the plasma allows Tg-gold (22) to bind to both immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b) , giving two red gold lines (23, 24) m addition to control line (25) for Tg rabbit antibody (16), as illustrated m Figure 8a.
Step 5
The reaction m the presence of autoantibody Tg-AAbl to Tg in the plasma allows autoantibody Tg-AAbl the plasma to bind to Tg-gold (22) preventing Tg-gold (22) from binding to immobilised antibody Tg-Abl (15a) . No red gold line is seen at the site of immobilised antibody Tg-Abl and indicates the presence of autoantibody Tg-AAbl to Tg m the plasma. This reaction m the absence of autoantibody Tg- AAb2 to Tg in the plasma allows Tg-gold (22) to bind to immobilised antibody Tg-Ab2 (15b) and a red gold line (24) is seen m addition to control line (25) , as illustrated m Figure 8b.
Step 6
The reaction the presence of autoantibody Tg-AAb2 to Tg m the plasma allows autoantibody Tg-AAb2 m the plasma to bind to Tg-gold (22 ) preventing Tg-gold (22 ) from binding to immobilised antibody Tg-Ab2 ( 15b) . No red gold line is seen at the site of immobilised antibody Tg-Ab2 (15b) and this indicates the presence of autoantibody Tg-AAb2 to Tg in the plasma. This reaction in the absence of autoantibody Tg-AAbl to Tg in the plasma allows Tg-gold (22) to bind to immobilised antibody Tg-Abl (15a) and a red gold line (23) is seen in addition to control line (25) , as illustrated in Figure 8c.
Step 7 The reaction in the presence of both autoantibodies Tg-AAbl and Tg-AAb2 to Tg in the plasma allows autoantibodies Tg- AAbl and Tg-AAb2 in the plasma to bind the Tg-gold (22) . Tg-gold (22) is prevented from binding to immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b) . No red gold lines are seen at the sites of immobilised antibodies Tg-Abl and Tg-Ab2 (15a, 15b), indicating the presence of autoantibodies Tg-AAbl and Tg-AAb2 to Tg in the plasma. Red gold control line (25) is seen, as illustrated in Figure 8d.
Referring to Figures 9a and 9b, there is shown a kit (26) for the screening of first and second autoantibodies to TPO
(autoantibodies TPO-AAbl and TPO-AAb2 respectively) . As previously referred to in kits (10, (13), (17), (18) and (21) , kit (26) comprises zone (2) for receiving a sample of body fluid, pad (3), nitrocellulose membrane (6) and wick
(8) . Non-immobilised first antibody to TPO, labelled with colloidal gold, (non-immobilised antibody TPO-Abl-gold)
(27) , is provided on pad (3) and is provided adjacent to zone (2) . Non-immobilised antibody TPO-Abl-gold (27) denotes non-immobilised antibody TPO-Abl previously labelled with -biotin-antibiotin-colloidal gold and subsequently applied to pad (3) . TPO (28) is dried to nitrocellulose membrane (6) . Purified second antibody to TPO (29) is immobilised (immobilised antibody TPO-Ab2) to nitrocellulose membrane (6) and is located downstream of non-immobilised antibody TPO-Abl-gold (27) and TPO (28) .
Non-immobilised antibody TPO-Abl-gold binds to the same site of TPO (28) as autoantibody TPO-AAbl. Immobilised antibody TPO-Ab2 binds to the same site of TPO (28) as autoantibody TPO-AAb2.
The following series of steps illustrate the use of kit (26) shown in Figures 9a and 9b in a screening method according to the present invention.
Step 1
The first step comprises applying a sample of blood or the like to receiving zone (2) . The sample will then flow towards wick (8) .
Step 2
The blood cells are retained in receiving zone (2) . The plasma then flows through pad (3) and forms a mixture with non-immobilised antibody TPO-Abl-gold (27) and this mixture flows towards TPO (28) .
Step 3 The mixture of step 2 reaches TPO ( 28 ) and TPO ( 28 ) also dissolves the mixture.
Step 4
Non- immobilised antibody TPO-Abl-gold (27) binds with TPO
(28) m the mixture, unless autoantibody TPO-AAbl is present m the plasma. In the presence of autoantibody TPO-AAbl the plasma, autoantibody TPO-AAbl and non- lmmobilised antibody TPO-Abl-gold (27) compete for binding with TPO (28) m the mixture.
Step 5
The mixture of plasma, non- immobilised antibody TPO-Abl- gold (27) and TPO (28) reach immobilised antibody TPO-Ab2
(29) .
Step 6
The reaction m the absence of autoantibodies TPO-AAbl and
TPO-AAb2 to TPO m the plasma allows TPO (28) to bind to non- immobilised antibody TPO-Abl-gold (27) and immobilised antibody TPO-Ab2 (29) . A red gold line (30) at the location of immobilised antibody TPO-Ab2 (29) indicates the absence of autoantibodies TPO-AAbl and TPO-AAb2 m the plasma, as illustrated m Figure 10a.
Step 7
The reaction the presence of autoantibody TPO-AAbl and / or autoantibody TPO-AAb2 m the plasma sets up a competition reaction wherein binding of autoantibodies TPO- AAbl and/or TPO-AAb2 competes w th binding of non- immobilised antibody TPO-Abl-gold (27) and immobilised antibody TPO-Ab2 (29) with TPO (28) . In the case where autoantibodies TPO-AAbl and / or TPO-AAb2 prevent binding of TPO (28) with non-immobilised antibody TPO-Abl-gold (27) and / or immobilised antibody TPO-Ab2 (29) , no red gold line is seen as illustrated m Figure 10b. Alternatively, where competition exists as above the presence of autoantibodies TPO-AAbl and TPO-AAb2, there may still be some binding of TPO (28) with non-immobilised antibody TPO- Abl-gold (27) and immobilised antibody TPO-Ab2 (29) , but such binding will be to a lesser extent compared to that of step 6, and a qualitative measure of the quantity of autoantibodies TPO-AAbl and TPO-AAb2 m the sample will be obtained.
The present invention will now be further illustrated by the following Examples which do not limit the scope of the invention m any way.
Example 1
The following tables give the results obtained using a TPO screening kit as shown in Figures la and lb and a Tg screening kit as shown in Figure 4.
Table 1 Patient sample results TPOAb TPOAb rapid assay
Figure imgf000040_0001
Table 2
Patient sample results - TgAb TgAb rapid assay
Figure imgf000041_0001
Example 2
This Example describes screening for autoantibodies to Tg using a kit as illustrated in Figures 7 and 8.
90 μl of plasma (or sera) or 30 μl of whole blood plus 60 μl of a diluent buffer (150 mM NaCl, 20 mM Tris pH7.6 ) were used. Results were obtained after 10 minutes. Prior art reference radioactive method was based on that of Beever et al Clinical Chemistry 35. (1989) 1949-1954. Table 3
(a) Results obtained in whole blood or plasma obtained from 30 healthy blood donors.
Figure imgf000042_0001
Table 4
(b) Results obtained with sera from patients with systemic lupus erythematosus
Figure imgf000043_0001
The above data shows that a method for screening Tg autoantibodies according to the present invention can detect Tg autoantibodies in plasma from healthy blood donors, or sera from patients with systemic lupus erythematosus, at the same prevalence as the reference radioactive test.
Example 3
The Example describes screening for autoantibodies to TPO using a kit as illustrated in Figures 9 and 10.
90 μl of plasma (or sera) or 30 μl of whole blood plus 60 μl of a diluent buffer (150 mM Nacl; 20 mM Tris pH 7.6) were used. Results were obtained after 10 minutes. Prior art reference radioactive method was based on that of Beever et al Clinical Chemistry 35 (1989) 1949-1954.
Table 5
(a) Results obtained in whole blood or plasma obtained from 30 healthy blood donors
Figure imgf000044_0001
Table 6
(b) Results obtained in sera from patients with suspected autoimmune thyroid disease
Figure imgf000045_0001
The above data indicates that a method for screening TPO autoantibodies according to the present invention can detect TPO autoantibodies m (a) healthy blood donors or
(b) patients suspected of having autoimmune thyroid disease at the same prevalence as the reference radioactive test.

Claims

CLAIMS :
1. A method of screening a sample of body fluid for at least one autoantibody to at least one antigen, which method comprises:
(a) providing a source of said at least one antigen to said autoantibody;
(b) providing a substrate having immobilised thereto at least one antibody to said antigen of step
(a) ;
(c) contacting said antigen source of step (a) with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present m said sample;
(d) allowing said mixture obtained m step (c) to flow relative to said substrate of step (b) so as to allow said mixture to contact said antibody immobilised to said substrate;
(e) providing labelling means so as to permit monitoring of binding of said autoantibody and said antigen present m said mixture obtained m step (c) ; and (f) monitoring said binding so as to provide an indication of the presence of said autoantibody m said sample of body fluid.
2. A method according to claim 1, wherein said antigen comprises a thyroid protein.
A method according to claim 2, wherein said thyroid protein is selected from the group consisting of thyroid peroxidase, thyroglobulm and thyroid stimulating hormone receptor.
A method according to claim 3, wherein said thyroid protein is selected from the group consisting of thyroid peroxidase and thyroglobulm.
5. A method of screening a sample of body fluid for at least one autoantibody to at least one antigen comprising a thyroid protein selected from the group consisting of thyroid peroxidase, thyroglobulm and thyroid stimulating hormone receptor, which method comprises :
(a) providing a source of said at least one antigen to said autoantibody; (b) providing a substrate having immobilised thereto at least one antibody to said antigen of step (a) ;
(c) contacting said antigen source of step (a) with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present m said sample;
(d) allowing said mixture obtained m step (c) to flow relative to said substrate of step (b) so as to allow said mixture to contact said antibody immobilised to said substrate;
(e) providing labelling means so as to permit monitoring of binding of said autoantibody and said antigen present m said mixture obtained in step (c) ; and
(f) monitoring said binding so as to provide an indication of the presence of said autoantibody m said sample of body fluid.
6. A method according to claim 5, wherein said thyroid protein is thyroid peroxidase or thyroglobulm.
7. A method according to any of claims 1 to 6 , which further comprises screening for the presence of at least one of thyroid stimulating hormone, thyroxme, tri-iodothyronme and thyroglobulm m said sample of body fluid.
8. A method according to any preceding claim, which comprises contacting step (c) said antigen source and said sample of body fluid with at least one substantially non- immobilised antibody to said antigen.
9. A method according to claim 8, wherein said non- lmmobilised antibody is provided substantially purified form.
10. A method according to claim 8 or 9 , wherein said non- immobilised antibody comprises a monoclonal antibody.
11. A method according to any of claims 8 to 10, wherein said non- immobilised antibody comprises an autoantibody to said antigen.
12. A method according to any preceding claim, wherein said monitoring m step (f) comprises observing a colorimetric change dependent on said binding of said autoantibody and said antigen present m said mixture of step (c) .
13. A method according to claim 12, wherein said labelling means include colloidal gold.
14. A method according to any preceding claim, which further comprises providing a positive control that is present m the presence or absence of the autoantibody or autoantibodies being screened.
15. A method according to any preceding claim, wherein said mixture obtained step (c) is allowed to flow along said substrate and interact with said antibody immobilised to said substrate.
16. A method according to claim 15, wherein at least said sample of body fluid is contacted with an application zone of said substrate, which application zone is provided upstream on said substrate relative to said immobilised antibody, and wherein said mixture is allowed to flow from said application zone along said substrate so as to interact with said immobilised antibody.
17. A method according to claim 16, wherein said application zone includes said source of said antigen of step (a) , and said mixture in step (c) is obtained by contacting said sample of body fluid with said antigen of said application zone.
18. A method according to claim 16 or 17 as dependent on any of claims 8 to 11, wherein said application zone further includes said non-immobilised antibody, and said mixture in step (c) is obtained by contacting said sample of body fluid and said antigen with said non-immobilised antibody present in said application zone .
19. A method according to claim 16, wherein said antigen source of step (a) and said sample of body fluid are contacted substantially remote from said substrate so as to provide said mixture of step (c) , and said mixture is subsequently contacted with said application zone.
20. A method according to claim 19 as dependent on any of claims 8 to 11, wherein said antigen source of step (a) , said sample of body fluid and/or said non- immobilised antibody are contacted substantially remote from said substrate so as to provide said mixture of step (c) , and said mixture is subsequently contacted with said application zone.
21. A method according to any preceding claim, wherein said substrate comprises a membrane of nitrocellulose, cellulose acetate or a polyamide .
22. A method according to any preceding claim, wherein said immobilised antibody is in substantially purified form.
23. A method according to any preceding claim, wherein said immobilised antibody comprises an autoantibody to said antigen.
24. A method according to any of claims 1 to 23, wherein said immobilised antibody comprises a monoclonal antibody.
25. A method according to any preceding claim, wherein said sample of body fluid comprises blood, plasma, serum or urine .
26. A method according to any preceding claim, which comprises screening said sample of body fluid for one said autoantibody.
27. A method according to claim 26, wherein said antigen includes a binding site to which either said autoantibody or said immobilised antibody can bind, whereby in step (d) binding of said immobilised antibody to said binding site is substantially precluded where said autoantibody has substantially bound to said binding site in step (c) .
28. A method according to any of claims 1 to 25, which comprises screening said sample of body fluid for at least first and second autoantibodies to said antigen, wherein at least first and second antibodies to said antigen are immobilised on said substrate in step (b) .
29. A method according to claim 28, wherein said antigen includes :
a first binding site to which either said first autoantibody or said first immobilised antibody can bind, whereby in step (d) binding of said first immobilised antibody to said first binding site is substantially precluded where said first autoantibody has substantially bound to said first binding site in step (c) ; and
a second binding site to which either said second autoantibody or said second immobilised antibody can bind, whereby in step (d) binding of said second immobilised antibody to said second binding site is substantially precluded where said second autoantibody has substantially bound to said second binding sice in step (c) ;
wherein said first and second binding sites are substantially distinct sites on said antigen.
30. A method according to any of claims 26 to 29, wherein said antigen is provided with said labelling means.
31. A method according to any of claims 26 to 30, as dependent on claim 14, wherein said positive control comprises attaching to the substrate at least one control antibody to the antigen, which control antibody binds to a site on the antigen distinct to a binding site thereof for the autoantibody or autoantibodies being screened.
32. A method according to any of claims 26 to 29 as dependent on any of claims 8 to 11, wherein said non- lmmobilised antibody is provided with said labelling means, which non- immobilised antibody is capable of binding to a site on said antigen substantially distinct from a binding site for either d) said autoantibody or autoantibodies being screened or (ii) said immobilised antibody, whereby m step (d) , antigen is allowed to be substantially bound both to said immobilised antibody and to said non- immobilised antibody.
33. A method according to any of claims 8 to 25 as dependent on any of claims 8 to 11, which comprises screening said sample of body fluid for at least first and second autoantibodies to said antigen, wherein said non- immobilised antibody is capable of binding to a site on said antigen to which either said first or second autoantibody can bind and which is substantially distinct to a binding site on said antigen for said immobilised antibody, whereby in step (d) antigen is allowed to be substantially bound both to said immobilised antibody and to said non- lmmobilised antibody.
34. A method according to claim 33, wherein said antigen includes :
a first binding site to which either said first autoantibody or said immobilised antibody can bind, whereby step (d) binding of immobilised antibody to said first binding site is substantially precluded where said first autoantibody has substantially bound to said first binding site m step (c) ; and
a second binding site to which either said second autoantibody or said non- immobilised antibody can bind;
wherein said first and second binding sites are substantially distinct sites on said antigen.
35. A method according to claim 33 or 34, wherein said non- immobilised antibody is provided with said labelling means.
36. A method according to claims 33 to 35 as dependent on claims 11 and 23, wherein said immobilised antibody comprises a first autoantibody to said antigen and said non-immobilised antibody comprises a second autoantibody to said antigen.
37. A method according to any of claims 32 to 36, wherein the positive control comprises attaching to the substrate at least one control agent that can bind to the at least one substantially non-immobilised antibody.
38. A kit for use in screening a sample of body fluid for at least one autoantibody to at least one antigen, which kit comprises:
(a) a source of said at least one antigen to said autoantibody;
(b) a substrate having immobilised thereto at least one antibody to said antigen;
(c) means for contacting said antigen source with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present in said sample;
(d) means for allowing said mixture to flow relative to said substrate so as to allow said mixture to contact said antibody immobilised to said substrate ; (e) labelling means to permit monitoring of binding of said autoantibody and said antigen present in said mixture; and (f) means for monitoring said binding so as to provide an indication of the presence of said autoantibody in said sample of body fluid.
39. A kit according to claim 38, wherein said antigen comprises a thyroid protein.
40. A kit according to claim 39, wherein said thyroid protein is selected from the group consisting of thyroid peroxidase, thyroglobulm and thyroid stimulating hormone receptor.
41. A kit according to claim 40, wherein said thyroid protein is selected from the group consisting of thyroid peroxidase and thyroglobulm.
42. A kit for use in screening a sample of body fluid for at least one autoantibody to at least one antigen comprising a thyroid protein selected from the group consisting of thyroid peroxidase, thyroglobulm and thyroid stimulating hormone receptor, which kit comprises :
(a) a source of said at least one antigen to said autoantibody;
(b) a substrate having immobilised thereto at least one antibody to said antigen; (c) means for contacting said antigen source with said sample of body fluid, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present m said sample; (d) means for allowing said mixture to flow relative to said substrate so as to allow said mixture to contact said antibody immobilised to said substrate ;
(e) labelling means to permit monitoring of binding of said autoantibody and said antigen present said mixture; and
(f) means for monitoring said binding so as to provide an indication of the presence of said autoantibody m said sample of body fluid.
43. A kit according to claim 42, wherein said thyroid protein is thyroid peroxidase or thyroglobulm.
44. A kit according to any of claims 38 to 43, which further comprises means for screening for the presence of at least one of thyroid stimulating hormone, thyroxme, tri-iodothyronme and thyroglobulm m said sample of body fluid.
45. A kit according to any of claims 38 to 44, which further comprises a source of at least one substantially non- immobilised antibody to said antigen and means whereby said non- immobilised antibody can be contacted with said antigen source and said sample of body fluid.
46. A kit according to claim 45, wherein said non- lmmobilised antibody is provided in substantially purified form.
47. A kit according to claim 45 or 46, wherein said non- lmmobilised antibody comprises a monoclonal antibody.
48. A kit according to any of claims 45 to 47, wherein said non- immobilised antibody comprises an autoantibody to said antigen.
49. A kit according to any of claims 38 to 48, wherein said monitoring means comprise means for observing a colorimetric change dependent on said binding of said autoantibody and said antigen present m said mixture.
50. A kit according to claim 49, wherein said labelling means include colloidal gold.
51. A kit according to any of claims 38 to 50, which further comprises a positive control that is present m the presence or absence of the autoantibody being screened.
52. A kit according to any of claims 38 to 51, wherein said substrate comprises an application zone for at least said sample of body fluid, which application zone is provideα upstream on said substrate relative to said immobilised antibody, whereby said mixture is allowed to flow from said application zone along said substrate so as to interact with said immobilised antibody.
53. A kit according to claim 52, wherein said application zone includes said source of said antigen, and said mixture is obtained by contacting said sample of body fluid with said antigen of said application zone.
54. A kit according to claim 52 or 53 as dependent on any of claims 45 to 48, wherein said application zone further includes said non-immobilised antibody, and means whereby said mixture is obtained by contacting said sample of body fluid and said antigen with said non-immobilised antibody present in said application zone .
55. A kit according to claim 52, wherein means are provided whereby said antigen source and said sample of body fluid are contacted substantially remote from said substrate so as to provide said mixture and means whereby said mixture is subsequently contacted with said application zone.
56. A kit according to claim 55 as dependent on any of claims 45 to 48, wherein means are provided whereby said antigen source, said sample of body fluid and/or said non-immobilised antibody are contacted substantially remote from said substrate so as to provide said mixture, and means whereby said mixture iΞ subsequently contacted with said application zone.
57. A kit according to any of claims 38 to 56, wherein said substrate comprises a membrane of nitrocellulose, cellulose acetate or a polyamide .
58. A kit according to any of claims 38 to 57, wherein said immobilised antibody is provided m substantially purified form.
59. A kit according to any of claims 38 to 58, wherein said immobilised antibody comprises an autoantibody to said antigen.
60. A kit according to any of claims 38 to 59, wherein said immobilised antibody comprises a monoclonal antibody.
61. A kit according to any of claims 38 to 60, wherein said sample of body fluid comprises blood, plasma, serum or urine .
62. A kit according to any of claims 38 to 61, for screening said sample of body fluid for one said autoantibody, wherein said antigen includes a binding site to which either said autoantibody or said immobilised antibody can bind, whereby binding of said immobilised antibody to said binding site is substantially precluded where said autoantibody has previously substantially bound to said binding site.
63. A kit according to any of claims 38 to 61, for screening said sample of body fluid for at least first and second autoantibodies to said antigen, wherein at least first and second antibodies to said antigen are immobilised on said substrate.
64. A kit according to claim 63, wherein said antigen includes :
a first binding site to which either said first autoantibody or said first immobilised antibody can bind, whereby binding of said first immobilised antibody to said first binding site is substantially precluded where said first autoantibody has previously substantially bound to said first binding site; and
a second binding site to which either said second autoantibody or said second immobilised antibody can bind, whereby binding of said second immobilised antibody to said second binding site is substantially precluded where said second autoantibody has previously substantially bound to said second binding site;
wherein said first and second binding sites are substantially distinct sites on the antigen.
65. A kit according to any of claims 61 to 64, wherein said antigen is provided with said labelling means.
66. A kit according to any of claims 62 to 65, as dependent on claim 51, wherein the positive control comprises attaching to the substrate at least one control antibody to the antigen, which control antibody binds to a site on the antigen distinct to a binding site thereof for the autoantibody or autoantibodies being screened.
67. A kit according to any of claims 62 to 64, as dependent on any of claims 45 to 48, wherein said non- immobilised antibody is provided with said labelling means, which non-immobilised antibody is capable of binding to a site on said antigen substantially distinct from a binding site for either (i) said autoantibody or autoantibodies being screened or (ii) said immobilised antibody, whereby antigen is allowed to be substantially bound both to said immobilised antibody and to said non-immobilised antibody.
68. A kit according to any of claims 38 to 61, as dependent on any of claims 45 to 48 for screening said sample of body fluid for at least first and second autoantibodies to said antigen, wherein said non- immobilised antibody is capable of binding to a site on said antigen to which either said first or second autoantibody can bind and which is substantially distinct to a binding site on said antigen for said immobilised antibody, whereby antigen is allowed to be substantially bound both to said immobilised antibody and to said non-immobilised antibody.
69. A kit according to claim 68, wherein said antigen includes:
a first binding site to which either said first autoantibody or said immobilised antibody can bind, whereby binding of immobilised antibody to said first binding site is substantially precluded where said first autoantibody has previously substantially bound to said first binding site; and
a second binding site to which either said second autoantibody or said non-immobilised antibody can bind;
wherein said first and second binding sites are substantially distinct sites on said antigen.
70. A kit according to claim 68 or 69, wherein said non- immobilised antibody is provided with said labelling means .
71. A kit according to any of claims 68 to 70, as dependent on claims 48 and 59, wherein said immobilised antibody comprises a first autoantibody to said antigen and said non-immobilised antibody comprises a second autoantibody to said antigen.
72. A kit according to any of claims 67 to 71, as dependent on claim 51, wherein the positive control comprises attaching to the substrate at least one control agent that can bind to the at least one substantially non-immobilised antibody.
73. Use of a kit according to any of claims 38 to 72, in screening a sample of body fluid for at least one autoantibody to at least one antigen.
74. A method of screening a patient for at least one autoantibody to at least one antigen, which method comprises :
(a) obtaining a sample of body fluid from said patient ;
(b) contacting said sample of body fluid of step (a) with an antigen source of a kit according to any of claims 38 to 72, so as to obtain a mixture wherein said antigen is allowed to substantially bind with said autoantibody, when the latter is present in said sample;
(c) allowing said mixture to flow relative to a substrate of a kit according to any of claims 38 to 72, so as to allow said mixture to contact said antibody immobilised to said substrate; and
(d) monitoring binding of said autoantibody and said antigen present in said mixture, so as to provide an indication of the presence of said autoantibody in said sample of body fluid from said patient .
75. A method according to claim 74, for testing said patient for an autoimmune thyroid disease.
76. A method according to claim 74 or 75, which further comprises screening for the presence of at least one of thyroid stimulating hormone, thyroxine, tri- iodothyronine and thyroglobulm in said sample of body fluid.
77. A method of treating a patient suffering from, or susceptible to, an autoimmune disease, which method comprises:
screening said patient for at least one autoantibody to at least one antigen as defined in any of claims 74 to 76; and
when at least one autoantibody is detected in a sample of body fluid obtained from said patient at a level indicative of an autoimmune disease, administering to said patient at least one therapeutically active substance effective in the treatment of the autoimmune disease .
78. In combination, a kit as defined in any of claims 38 to 72, and at least one therapeutically active substance effective in the treatment of an autoimmune disease .
79. A method substantially as hereinbefore described, substantially as described in one of the Examples.
80. A kit substantially as hereinbefore described, substantially as described in one of the Examples.
81. A kit substantially as hereinbefore described, substantially as illustrated in one or more of Figures la, lb, 2a, 2b, 3a, 3b, 4, 5, 6, 7a, 7b, 8a, 8b, 8c, 8d, 9a, 9b, 10a or 10b.
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US7001775B1 (en) 2006-02-21
ATE323286T1 (en) 2006-04-15
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