EP2201130A1 - Procédés sensibles et rapides d'utilisation de récepteurs chimériques pour identifier une maladie auto-immune - Google Patents

Procédés sensibles et rapides d'utilisation de récepteurs chimériques pour identifier une maladie auto-immune

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Publication number
EP2201130A1
EP2201130A1 EP08835584A EP08835584A EP2201130A1 EP 2201130 A1 EP2201130 A1 EP 2201130A1 EP 08835584 A EP08835584 A EP 08835584A EP 08835584 A EP08835584 A EP 08835584A EP 2201130 A1 EP2201130 A1 EP 2201130A1
Authority
EP
European Patent Office
Prior art keywords
cells
cho
cell
tsh
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08835584A
Other languages
German (de)
English (en)
Other versions
EP2201130A4 (fr
Inventor
James L. Brown
Leonard Kohn
David Scholl
Yunsheng Li
Giorgio Napolitano
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diagnostic Hybrids Inc
Original Assignee
Diagnostic Hybrids Inc
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
Priority claimed from US11/906,189 external-priority patent/US8293879B2/en
Application filed by Diagnostic Hybrids Inc filed Critical Diagnostic Hybrids Inc
Publication of EP2201130A1 publication Critical patent/EP2201130A1/fr
Publication of EP2201130A4 publication Critical patent/EP2201130A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/046—Thyroid disorders

Definitions

  • the present invention provides methods and compositions useful in the diagnosis of autoimmune diseases.
  • the present invention provides methods and compositions for use in the diagnosis and management of Graves' disease.
  • one composition comprises a chimeric thyroid stimulating hormone receptor having improved sensitivity and specificty for circulating thyroid stimulating immunoglobulin. Assays using such chimeric receptors can be optimized in the presence of a glucocorticoid.
  • Graves' disease also referred to as "diffuse toxic goiter"
  • Graves' disease is the leading cause of hyperthyroidism due to the action of autoantibodies that recognize and bind to receptors present on the thyroid gland, resulting in gland growth and over-production of thyroid hormone. Graves' disease is reported to be the most frequent cause of hyperthyroidism in childhood and adolescence (See, Boter and Brown, J. Pediatr. 132:612-618 (1998)).
  • the present invention provides methods and compositions useful in the diagnosis and management of autoimmune diseases.
  • the present invention provides methods and compositions for the diagnosis and management of Graves' disease.
  • one composition comprises a chimeric thyroid stimulating hormone receptor having improved sensitivity and specificty for circulating thyroid stimulating immunoglobulin. Assays using such chimeric receptors can be optimized in the presence of a glucocorticoid.
  • the present invention contemplates a method, comprising: a) providing; i) a cell line comprising a stably transfected vector encoding a chimeric TSH receptor and a luciferase gene; ii) a serum sample derived from a patient suspected of having Graves' disease; and iii) a cell culture medium comprising a glucocorticoid; b) contacting the serum sample with the cell line and the medium under conditions such that the luciferase gene emits a detectable signal.
  • the method further comprises step c) measuring the signal intensity, wherein the intensity correlates with a thyrotropin stimulating hormone receptor autoantibody concentration present in the sample.
  • the glucocorticoid is selected from the group comprising dexamethasone, prednisone, hydrocortisone, fluticasone, or cortisone.
  • the contacting further comprises polyethylene glycol.
  • the chimeric TSH receptor comprises an amino acid sequence derived from rat chorionic hormone gonadotropin receptor.
  • the amino acid sequence comprises seventy three amino acids corresponding to amino acid residues 262-335 of a human TSH receptor amino acid sequence.
  • the serum sample comprises TSH receptor autoantibodies.
  • the autoantibodies comprise TSH stimulating autoantibodies.
  • the autoantibodies comprise TSH blocking antibodies.
  • the present invention contemplates a kit comprising a chimeric TSH receptor and a luciferin-luciferase system capable of detecting serum TSH autoantibodies, wherein the system comprises a glucocorticoid.
  • the glucocorticoid is selected from the group comprising dexamethasone, prednisone, hydrocortisone, fluticasone, or cortisone.
  • the receptor comprises a human TSH receptor amino acid sequence.
  • the receptor comprises a rat chorionic hormone receptor amino acid sequence.
  • the rat receptor amino acid sequence comprises amino acid residues 262-335.
  • the kit further comprises a cell line capable of expressing the chimeric TSH receptor and the luciferin-luciferase system. In one embodiment, the kit further comprises polyethylene glycol. In one embodiment, the kit comprises a vector encoding the chimeric TSH receptor and a luciferase gene. In one embodiment, the vector further comprises a promoter in operably linked to the vector. In one embodiment, the promoter comprises a glycoprotein alpha subunit promoter. In one embodiment, the cell line comprises CHO cells. In one embodiment, the cell line comprises RD cells. In one embodiment, the kit further comprises an instruction sheet.
  • the present invention provides methods for determining the presence of thyroid-stimulating autoantibodies in a test sample, comprising: a) providing i) a test sample suspected of containing thyroid-stimulating autoantibodies, ii) cultured cells comprising a glucocorticoid contained within a testing means, wherein the cells express a chimeric TSH receptor and a luciferin-luciferase system, and iii) polyethylene glycol; b) exposing the test sample to the cultured cells and polyethylene glycol under conditions such that thyroid-stimulating antibodies are detectable using a luciferin- luciferase system; and c) observing for the presence of detectable thyroid-stimulating antibodies.
  • the glucocorticoid is selected from the group comprising dexamethasone, prednisone, hydrocortisone, fluticasone, or cortisone.
  • the cultured cells are selected from the group consisting of RDluc and CHORluc cells.
  • the observing is conducted using a luminometer.
  • the cAMP concentration is determined by the luciferin-luciferase system.
  • the methods further comprise a Growth Medium, while in other embodiments, the methods further comprise a Stimulation Medium.
  • the cultured cells are exposed to the Growth Medium prior to exposure of the test sample.
  • the cultured cells are exposed to Stimulation Medium containing the test sample.
  • the Stimulation Medium comprises polyethylene glycol.
  • the present invention also provides methods for determining the presence of thyroid-stimulating autoantibodies in a test sample, comprising: a) providing; i) a test sample suspected of containing thyroid-stimulating autoantibodies, ii) cultured cells comprising a glucocorticoid, wherein the cells are selected from the group comprising RD-Rluc or CHO-Rluc cells contained within a testing means, wherein the cells express a chimeric TSH receptor, and iii) polyethylene glycol; b) exposing the test sample to the cultured cells and the polyethylene glycol under conditions such that thyroid stimulating antibodies are detectable using a luciferin-luciferase system; and c) observing for the presence of detectable thyroid-stimulating antibodies, wherein observing is conducted using a luminometer.
  • the glucocorticoid is selected from the group comprising dexamethasone, prednisone, hydrocortisone, fluticasone, or cortisone.
  • the cAMP concentration is determined by the luciferin-luciferase system.
  • the methods further comprise a Growth Medium, while in other embodiments the methods further comprise a Stimulation Medium.
  • the cultured cells are exposed to the Growth Medium prior to exposure of the test sample.
  • the cultured cells are exposed to the Stimulation Medium containing the test sample.
  • the Stimulation Medium comprises polyethylene glycol.
  • the present invention also provides methods for determining the presence of thyroid-stimulating autoantibodies in a test sample, comprising: a) providing; i) a test sample suspected of containing thyroid-stimulating autoantibodies, ii) cultured cells comprising a glucocorticoid, wherein the cells are selected from the group comprising RD-Rluc or CHO-Rluc cells contained within a testing means, wherein the cells express a chimeric TSH receptor, iii) Growth Medium, and iv) Stimulation Medium, wherein the Stimulation Medium comprises polyethylene glycol; b) exposing the cultured cells to Growth Medium to produce grown cells; c) exposing the test sample to the grown cells and Stimulation Medium under conditions such that thyroid-stimulating antibodies are detectable using the luciferin-luciferase system; and d) observing for the presence of detectable thyroid-stimulating antibodies, wherein said observing is conducted using a luminometer.
  • the glucocorticoid is selected from the group comprising dexamethasone, prednisone, hydrocortisone, fluticasone, or cortisone.
  • the cAMP concentration is determined by the luciferase-luciferin system.
  • Figure 1 provides results for serial 3-fold dilutions of three Graves' disease IgG samples (from untreated Graves' patients), in assays utilizing Stimulation Medium containing 6% PEG-8000.
  • Figure 2 provides a comparison of CHO-Rluc luciferase results with the FRTL-5 c AMP results for IgGs from 35 untreated Graves' patients.
  • Figure 3 provides a comparison of CHO-Rluc luciferase results with CHO-R cAMP results for IgGs from 35 untreated Graves' patients.
  • Figure 4 provides a comparison of CHO-R cAMP results with FRTL-5 cAMP results for IgGs from 35 untreated Graves' patients.
  • Figure 5 shows the linearity of the response to bTSH of the CHO-Rluc cells.
  • Figure 6 shows the results for a group of samples with known TSI results using
  • FRTL-5 cells (10 ⁇ l samples of LCA TSI specimens).
  • Figure 7 shows the results for a group of normal samples (10 ⁇ l of AML "normal" specimens).
  • Figure 8 shows one embodiment of a DNA sequence for a chimeric hTSH/mLH (Mc4) receptor comprising 2,324 base pairs and encoding 730 amino acids.
  • the underlined letters are the human TSHR sequence.
  • the letters in italics are the rat LHR sequence.
  • "* " (T) in the rat LHR sequence is a G in the wild type sequence. This G to T mutation resulted in an amino acid change from Arginine to Serine.
  • Figure 9 shows one embodiment of a 236 nucleotide glycoprotein alpha subunit promoter comprising a cyclic AMP (cAMP) regulatory element (CRE) (AF401991) sequence alignment with a GPH promoter amplified by PCR from HEK cells. Shaded areas indicate homology. Non-highlighted areas designate the flanking region of the promoter in the plasmid.
  • cAMP cyclic AMP
  • CRE regulatory element
  • Figure 10 presents exemplary data showing the response of the CH0-RMc4, RD- RMc4 and CHO-RLuc cell lines to negative and positive TSI-containing sera.
  • Figure 1OA Luciferase assay on CHO-RLuc and CHO-RMc4 cell lines induced with TSI negative and positive sera.
  • Figure 1OB The ratio of S/N derived from the luciferase assay on CHO-RLuc and CHO-RMc4 cell lines induced with TSI negative and positive sera.
  • Figure 1OC Luciferase assay on CHO-RLuc and RD-RMc4 cell lines induced with TSI negative and positive sera.
  • Figure 10D The ratio of S/N derived from the luciferase assay on CHO-RLuc and RD-RMc4 cell lines induced with TSI negative and positive sera.
  • Figure 1OE The ratio of S/N derived from the luciferase assay on CHO-RLuc, CHO-RMc4 and RD-RMc4 cell lines induced with TSI negative and positive sera.
  • Figure 11 presents exemplary data showing signal-to-noise (S/N) ratios for RD- RMc4 and CHO-RLuc cell lines in response to a serum dilution profile.
  • Figure 1 IA The S/N ratio from the luciferase assay on CHO-RLuc and RD-
  • Figure HB The S/N ratio from the luciferase assay on CHO-RMc4 cell line induced with dilution of the TSI positive serum.
  • Figure 11C The S/N ratio from the luciferase assay on CHO-RMc4 cell line induced with higher dilutions of the TSI positive
  • Figure 12 presents exemplary data comparing TSH sensitivity between a CHO- RMc4 cell line and a CHO-RLuc cell line.
  • Figure 13 presents exemplary data presenting the distribution of signal-to-noise ratios from human sera using CHO-RMc4 and CHO-RLuc cell lines.
  • Figure 14 presents exemplary data showing the relative sensitivity of the CHO- RMc4, RD-RMc4 and CHO-RLuc cell lines to clinical patient serum samples.
  • Figure 15 presents exemplary amino acid sequences: lutenizing hormone receptor:
  • FIG. 15 A Callithrix jacchus (white-tufted-ear marmoset) CAJ57370
  • Figure 15B Coturnix japonica (Japanese quail) AAB32614
  • FIG. 15C Gallus gallus (chicken) NP_990267
  • Figure 15D Mus musculus (mouse) AAB24402
  • Figure 15E Bos taurus (cow)
  • Figure 16 illustrates a representative arrangement of TSI samples in a testing plate.
  • Figure 17 present exemplary Relative Light Unit (RLU) data showing that the alternative glucocorticoids fluticasone, prednisone, hydrocortisone and cortisone provide equal signal intensities of the CHO-RMc4 assay when compared to 40 ⁇ M dexamethasone.
  • RLU Relative Light Unit
  • Figure 18 present exemplary Serum Reference Unit percentages (SSR%) data showing that the alternative glucocorticoids fluticasone, prednisone, hydrocortisone and cortisone provide an improved CHO-RMc4 assay.
  • SSR% Serum Reference Unit percentages
  • sample and “specimen” in the present specification and claims are used in their broadest sense. On the one hand, they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompass all types of samples obtained from humans and other animals, including but not limited to, body fluids (e.g., blood), as well as solid tissue.
  • body fluids e.g., blood
  • Biological samples may be animal, including human, fluid or tissue, food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • kit is used in reference to a combination of reagents and other materials.
  • the term "antibody” is used in reference to any immunoglobulin molecule that reacts with a specific antigen. It is intended that the term encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents, non-human primates, caprines, bovines, equines, ovines, etc.).
  • immunoglobulin e.g., IgG, IgM, IgA, IgE, IgD, etc.
  • the term "antigen” is used in reference to any substance that is capable of reacting with an antibody. It is intended that this term encompass any antigen and "immunogen” (i.e., a substance which induces the formation of antibodies). Thus, in an immunogenic reaction, antibodies are produced in response to the presence of an antigen (immunogen) or portion of an antigen.
  • antigen fragment and “portion of an antigen” are used in reference to a portion of an antigen. Antigen fragments or portions may occur in various sizes, ranging from a small percentage of the entire antigen to a large percentage, but not 100% of the antigen.
  • antigen fragments or portions may, but are not required to comprise an "epitope" recognized by an antibody.
  • Antigen fragments or portions also may or may not be immunogenic.
  • autoantibodies refers to antibodies that are capable of reacting against an antigenic constituent of an individual's own tissue or cells (e.g., the antibodies recognize and bind to "self antigens).
  • immunoassay is used in reference to any method in which antibodies are used in the detection of an antigen. It is contemplated that a range of immunoassay formats be encompassed by this definition, including but not limited to, direct immunoassays, indirect immunoassays, and “sandwich” immunoassays.” However, it is not intended that the present invention be limited to any particular format. It is contemplated that other formats, including radioimmunoassays (RIA), immunofluorescent assays (IFA), and other assay formats, including, but not limited to, variations on the ELISA, RIA and/or IFA methods will be useful in the method of the present invention.
  • RIA radioimmunoassays
  • IFA immunofluorescent assays
  • the term "capture antibody” refers to an antibody that is used to bind an antigen and thereby permit the recognition of the antigen by a subsequently applied antibody.
  • the capture antibody may be bound to a microtiter well and serve to bind an antigen of interest present in a sample added to the well.
  • Another antibody (termed the “primary antibody”) is then used to bind to the antigen-antibody complex, in effect to form a "sandwich” comprised of antibody-antigen-antibody complex. Detection of this complex can be performed by several methods.
  • the primary antibody may be prepared with a label such as biotin, an enzyme, a fluorescent marker, or radioactivity, and may be detected directly using this label.
  • a labelled "secondary antibody” or “reporter antibody” which recognizes the primary antibody may be added, forming a complex comprised of an antibody-antigen-antibody-antibody complex. Again, appropriate reporter reagents are then added to detect the labelled antibody. Any number of additional antibodies may be added as desired. These antibodies may also be labelled with a marker, including, but not limited to an enzyme, fluorescent marker, or radioactivity.
  • reporter reagent or “reporter molecule” is used in reference to compounds which are capable of detecting the presence of antibody bound to antigen.
  • a reporter reagent may be a colorimetric substance attached to an enzymatic substrate.
  • reporter reagents include, but are not limited to, fluorogenic and radioactive compounds or molecules.
  • This definition also encompasses the use of biotin and avidin-based compounds (e.g., including, but not limited to, neutravidin and streptavidin) as part of the detection system.
  • biotinylated antibodies may be used in the present invention in conjunction with avidin-coated solid support.
  • signal is used in reference to an indicator that a reaction has occurred, for example, binding of antibody to antigen.
  • signals in the form of radioactivity, fluorogenic reactions, luminscent and enzymatic reactions will be used with the present invention.
  • the signal may be assessed quantitatively as well as qualitatively.
  • the term "signal intensity" refers to magnitude of the signal strength wherein the intensity correlates with the amount of reaction substrate.
  • a luciferin-luciferase system generates a signal intensity that correlates with the amount of cAMP generated by thyrotropin stimulating hormone receptor autoantibodies.
  • luciferin-luciferase system refers to any process or method that allows the contact of luciferin and luciferase in the presence of a substrate (i.e., for example, cAMP) under conditions such that the resulting luminesence may be detected.
  • a substrate i.e., for example, cAMP
  • Such a system may be comprised within a transfected host cell encoded by a vector, or provided in separate kit containers whereby the contents may be mixed together.
  • solid support is used in reference to any solid material to which reagents such as antibodies, antigens, and other compounds may be attached.
  • solid supports include microscope slides, coverslips, beads, particles, cell culture flasks, as well as many other items.
  • cell staining is used in reference to methods used to label or stain cells to enhance their visualization. This staining or labelling may be achieved through the use of various compounds, including but not limited to, fluorochromes, enzymes, gold, and iodine. It is contemplated that the definition encompasses such methods as "in situ chromogenic assays," in which a test (i.e., an assay) is conducted on a sample in situ. It is also contemplated that the in situ chrdmogenic assay will involve the use of an immunoassay (i.e., an ELISA).
  • an immunoassay i.e., an ELISA
  • the term “Growth Medium” refers to a culture medium formulated to contain various growth factors including, but not limited to, vitamins, amino acids, co- factors, and any other appropriate nutrients to enhance growth and replication of cells in culture.
  • the term “Stimulation Medium” refers to a medium formulated to be deficient in certain constituents (e.g., sodium chloride), in order to enhance the stimulation of by TSH and/or TSI, thereby increasing the resulting signal (e.g., cAMP and/or luciferase).
  • the term "Starvation Medium” refers to a medium formulated to be deficient in at least one growth factors included in the Growth Medium. In preferred embodiments, this medium contains only the salts and glucose necessary to sustain cells for a short period of time.
  • organism and “microorganism,” are used to refer to any species or type of microorganism, including but not limited to viruses and bacteria, including rickettsia and chlamydia. Thus, the term encompasses, but is not limited to DNA and RNA viruses, as well as organisms within the orders Rickettsiales and Chlamydiales.
  • culture refers to any sample or specimen which is suspected of containing one or more microorganisms.
  • Purure cultures are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to “mixed cultures,” which are cultures in which more than one genus and/or species of microorganism are present.
  • cell type refers to any cell, regardless of its source or characteristics.
  • cell line refers to cells that are cultured in vitro, including primary cell lines, finite cell lines, continuous cell lines, and transformed cell lines.
  • primary cell culture refers to cell cultures that have been directly obtained from animal or insect tissue. These cultures may be derived from adults as well as fetal tissue.
  • the term "finite cell lines,” refer to cell cultures that are capable of a limited number of population doublings prior to senescence.
  • continuous cell lines refer to cell cultures that have undergone a "crisis” phase during which a population of cells in a primary or finite cell line apparently ceases to grow, but yet a population of cells emerges with the general characteristics of a reduced cell size, higher growth rate, higher cloning efficiency, increased tumorigenicity, and a variable chromosomal complement. These cells often result from spontaneous transformation in vitro. These cells have an indefinite lifespan.
  • transformed cell lines refers to cell cultures that have been transformed into continuous cell lines with the characteristics as described above.
  • Transformed cell lines can be derived directly from tumor tissue and also by in vitro transformation of cells with whole virus (e.g., SV40 or EBV), or DNA fragments derived from a transforming virus using vector systems.
  • whole virus e.g., SV40 or EBV
  • hybridomas refers to cells produced by fusing two cell types together. Commonly used hybridomas include those created by the fusion of antibody-secreting B cells from an immunized animal, with a malignant myeloma cell line capable of indefinite growth in vitro. These cells are cloned and used to prepare monoclonal antibodies.
  • the term "mixed cell culture,” refers to a mixture of two types of cells.
  • the cells are cell lines that are not genetically engineered, while in other preferred embodiments the cells are genetically engineered cell lines.
  • the one or more of the cell types is "permissive" (i.e., virus is capable of replication and spread from cell to cell within the culture).
  • the present invention encompasses any combination of cell types suitable for the detection, identification, and/or quantitation of viruses in samples, including mixed cell cultures in which all of the cell types used are not genetically engineered, mixtures in which one or more of the cell types are genetically engineered and the remaining cell types are not genetically engineered, and mixtures in which all of the cell types are genetically engineered.
  • the term "suitable for the detection of intracellular parasites,” refers to cell cultures that can be successfully used to detect the presence of an intracellular parasite in a sample. In preferred embodiments, the cell cultures are capable of maintaining their susceptibility to infection and/or support replication of the intracellular parasite. It is not intended that the present invention be limited to a particular cell type or intracellular parasite. As used herein, the term “susceptible to infection” refers to the ability of a cell to become infected with virus or another intracellular organism.
  • permissive infections it is not intended that the term be so limited, as it is intended that the term encompass circumstances in which a cell is infected, but the organism does not necessarily replicate and/or spread from the infected cell to other cells.
  • viral proliferation describes the spread or passage of infectious virus from a permissive cell type to additional cells of either a permissive or susceptible character.
  • the terms “monolayer,” “monolayer culture,” and “monolayer cell culture,” refer to cells that have adhered to a substrate and grow as a layer that is one cell in thickness.
  • Monolayers may be grown in various vessels including, but not limited to, flasks, tubes, coverslips (e.g., shell vials), roller bottles, etc. Cells may also be grown attached to microca ⁇ ers, including but not limited to beads.
  • the term "suspension,” and “suspension culture,” refers to cells that survive and proliferate without being attached to a substrate. Suspension cultures are typically produced using hematopoietic cells, transformed cell lines, and cells from malignant tumors.
  • the terms "culture media,” and “cell culture media,” refers to media that are suitable to support the growth of cells in vitro (i.e., cell cultures). It is not intended that the term be limited to any particular culture medium. For example, it is intended that the definition encompass outgrowth as well as maintenance media. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures of interest.
  • intracellular parasite refers to any organism which requires an intracellular environment for its survival and/or replication.
  • Obligate intracellular parasites include viruses, as well as many other organisms, including certain bacteria including, but not limited to, most members of the orders: i) Rickettsiales: for example, Coxiella, Rickettsia and Ehrlichia; and ii) Chlamydiales: for example, C. trachomatis, C. psittaci.
  • intracellular parasite refers to any organism that may be found within the cells of a host animal, including but not limited to obligate intracellular parasites briefly described above.
  • intracellular parasites include organisms such as Brucella, Listeria, Mycobacterium (e.g., M. tuberculosis and M. leprae), and Plasmodium, as well as Rochalirnea.
  • antimicrobial is used in reference to any compound which inhibits the growth of, or kills microorganisms. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as antibiotics which are produced naturally or synthetically. It is also intended that the term includes compounds and elements that are useful for inhibiting the growth of, or killing microorganisms.
  • chromogenic compound and “chromogenic substrate,” refer to any compound useful in detection systems by their light absorption or emission characteristics. The term is intended to encompass any enzymatic cleavage products, soluble, as well as insoluble, which are detectable either visually or with optical machinery. Included within the designation “chromogenic” are all enzymatic substrates which produce an end product which is detectable as a color change.
  • colors such as indigo, blue, red, yellow, green, orange, brown, etc.
  • fluorochromic or fluorogenic compounds which produce colors detectable with fluorescence (e.g., the yellow-green of fluorescein, the red of rhodamine, etc.). It is intended that such other indicators as dyes (e.g., pH) and luminogenic compounds be encompassed within this definition.
  • pH indicator encompasses all compounds commonly used for detection of pH changes, including, but not limited to phenol red, neutral red, bromthymol blue, bromcresol purple, bromcresol green, bromchlorophenol blue, m-cresol purple, thym.ol blue, bromcresol purple, xylenol blue, methyl red, methyl orange, and cresol red.
  • redox indicator and "oxidation-reduction indicator,” encompasses all compounds commonly used for detection of oxidation/reduction potentials (i.e., "eH”) including, but not limited to various types or forms of tetrazolium, resazurin, and methylene blue.
  • inoculating suspension or “inoculant,” is used in reference to a suspension which may be inoculated with organisms to be tested. It is not intended that the term “inoculating suspension,” be limited to a particular fluid or liquid substance.
  • inoculating suspensions may be comprised of water, saline, or an aqueous solution. It is also contemplated that an inoculating suspension may include a component to which water, saline or any aqueous material is added. It is contemplated in one embodiment, that the component comprises at least one component useful for the intended microorganism. It is not intended that the present invention be limited to a particular component.
  • the term “primary isolation,” refers to the process of culturing organisms directly from a sample.
  • the term “isolation,” refers to any cultivation of organisms, whether it be primary isolation or any subsequent cultivation, including “passage,” or “transfer,” of stock cultures of organisms for maintenance and/or use.
  • Presumptive diagnosis refers to a preliminary diagnosis which gives some guidance to the treating physician as to the etiologic organism involved in the patient's disease. Presumptive diagnoses are often based on “presumptive identifications,” which as used herein refer to the preliminary identification of a microorganism.
  • the term “definitive diagnosis” is used to refer to a final diagnosis in which the etiologic agent of the patient's disease has been identified.
  • the term “definitive identification” is used in reference to the final identification of an organism to the genus and/or species level.
  • the term “recombinant DNA molecule,” as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
  • DNA molecules are said to have "5 1 ends” and "3 1 ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides is referred to as the "5 1 end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
  • discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.
  • the promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region (enhancer elements can exert their effect even when located 3' of the promoter element and the coding region). Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
  • an oligonucleotide having a nucleotide sequence encoding a gene refers to a DNA sequence comprising the coding region of a gene or, in other words, the DNA sequence which encodes a gene product.
  • the coding region may be present in either a cDNA or genomic DNA form.
  • Suitable control elements such as enhancers, promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript.
  • the coding region utilized in the vectors of the present invention may contain endogenous enhancers and/or promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
  • transcription unit refers to the segment of DNA between the sites of initiation and termination of transcription and the regulatory elements necessary for the efficient initiation and termination.
  • a segment of DNA comprising an enhancer/promoter, a coding region, and a termination and polyadenylation sequence comprises a transcription unit.
  • regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
  • a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
  • Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (defined infra).
  • reporter gene construct refers to a recombinant DNA molecule containing a sequence encoding the product of a reporter gene and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • reporter gene refers to an oligonucleotide having a sequence encoding a gene product (typically an enzyme) which is easily and quantifiably assayed when a construct comprising the reporter gene sequence operably linked to a heterologous promoter and/or enhancer element is introduced into cells containing (or which can be made to contain) the factors necessary for the activation of the promoter and/or enhancer elements.
  • reporter genes include but are not limited to bacterial genes encoding ⁇ -galactosidase (lacZ, the bacterial chloramphenicol acetyltransferase (cat) genes, firefly luciferase genes and genes encoding ⁇ -glucuronidase (GUS).
  • Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses
  • promoters analogous control elements, i.e., promoters, are also found in prokaryotes.
  • the selection of a particular promoter and enhancer depends onwhat cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. ScL, 11 :287 (1986), and Maniatis, et al., supra (1987)).
  • the S V40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema, et al., EMBO J. 4:761 (1985)).
  • Two other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor l ⁇ gene (Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene 91:217 (1990); and Mizushima and Nagata, Nuc. Acids.
  • Rous sarcoma virus Rous sarcoma virus
  • human cytomegalovirus Boshart et aL, Cell 41 :521 (1985)
  • promoter/enhancer denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (for example, the long terminal repeats of retroviruses contain both promoter and enhancer functions).
  • the enhancer/promoter may be "endogenous,” or “exogenous,” or “heterologous.”
  • An endogenous enhancer/promoter is one which is naturally linked with a given gene in the genome.
  • An exogenous (heterologous) enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques).
  • Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8).
  • a commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.
  • Efficient expression of recombinant DNA sequences in eukaryotic cells requires signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.
  • the term "poly A site,” or "poly A sequence,” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
  • the poly A signal utilized in an expression vector may be "heterologous" or "endogenous.”
  • An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
  • a heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.
  • a commonly used heterologous poly A signal is the SV40 poly A signal.
  • the SV40 poly A signal is contained on a 237 bp BamHI/BcII restriction fragment and directs both termination and polyadenylation (Sambrook, supra, at 16.6-16.7). This 237 bp fragment is contained within a 671 bp BamHI/Pstl restriction fragment.
  • the term "genetically engineered cell line,” refers to a cell line that contains heterologous DNA introduced into the cell line by means of molecular biological techniques (i.e., recombinant DNA technology).
  • the term "vector” as used herein, refers to a nucleotide sequence comprising at least a promoter and a gene of interest. Such a gene of interest may encode an amino acid sequence for the purpose of expressing the amino acid sequence (i.e., for example, a TSH receptor amino acid sequence).
  • a vector has the capability of becoming integrated into foreign DNA to form a stable transfected cell.
  • stable trans fee tion or “stably transfected,” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell which has stably integrated foreign DNA into the genomic DNA.
  • stable transfection refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell which has stably integrated foreign DNA into the genomic DNA.
  • RDluc refers to an RD cell line having been stably transfected with a luciferase gene.
  • RD-Rluc refers to a RD cell line having been stably transfected with a luciferase gene and that displays an exogenous receptor (i.e., for example, a TSH receptor including but not limited to, a Mc4 receptor).
  • CHOluc refers to a CHO cell line having been stably transfected with a luciferase gene.
  • CHO-Rluc refers to a CHO cell line having been stably transfected with a luciferase gene and that displays an exogenous receptor (i.e., for example, a TSH receptor including but not limited to, a wild-type receptor).
  • CH0-RMc41uc refers to a CHO cell line having been stably trasnfected with a luciferase gene that displays a chimeric receptor, (i.e., for example, a TSH receptor that comprises amino acid sequences derived from a rat chorionic gonadotrophin receptor).
  • selectable marker refers to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
  • Selectable markers may be "dominant”; a dominant selectable marker encodes an enzymatic activity which can be detected in any mammalian cell line.
  • dominant selectable markers examples include the bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid.
  • Other selectable markers are not dominant in that their use must be in conjunction with a cell line that lacks the relevant enzyme activity.
  • non-dominant selectable markers examples include the thymidine kinase (tk) gene which is used in conjunction with tk cell lines, the CAD gene which is used in conjunction with CAD-deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is used in conjunction with hprt cell lines.
  • tk thymidine kinase
  • CAD CAD-deficient cells
  • hprt mammalian hypoxanthine-guanine phosphoribosyl transferase
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • confluent or “confluency” as used herein in reference to an adherent cell line define a condition wherein cells throughout a culture are in contact with each other creating what appears to be a continuous sheet or "monolayer" of cells.
  • CPE cytopathic effect
  • changes in cellular structure i.e., a pathologic effect
  • Common cytopathic effects include cell destruction, syncytia (i.e., fused giant cells) formation, cell rounding vacuole formation, and formation of inclusion bodies.
  • CPE results from actions of a virus on permissive cells that negatively affect the ability of the permissive cellular host to preform its required functions to remain viable.
  • CPE is evident when cells, as part of a confluent monolayer, show regions of non-confluence after contact with a specimen that contains a virus.
  • the observed microscopic effect is generally focal in nature and the foci is initiated by a single virion.
  • CPE may be observed throughout the monolayer after a sufficient period of incubation.
  • Cells demonstrating viral induced CPE usually change morphology to a rounded shape, and over a prolonged period of time can die and be released form their anchorage points in the monolayer.
  • the area is called a viral plaque, which appears as a hole in the monolayer. Cytopathic effects are readily discernable and distinguishable by those skilled in the art.
  • the abbreviation "ONPG,” represents o-Nitrophenyl-13-D-Galactopyranoside.
  • ONPG is a substrate for the enzyme ⁇ -galactosidase ( ⁇ -gal).
  • ⁇ -gal ⁇ -galactosidase
  • X-gal represents the chemical compound 5-bromo-4-chloro- 3-indolyl-(3-D-galactopyranoside, a substrate for the enzyme ⁇ -galactosidase.
  • the reaction between X-gal and ⁇ -galactosidase results in the formation of a blue precipitate which is visually discernable.
  • hybridization is the annealing of two complementary DNA molecules whose base sequences match according to the rules of base pairing. DNA hybridization is used to identify or quantify an unknown or "target” DNA by hybridization to a known DNA or "probe.”
  • the probe is typically labeled with a reporter molecule such as 125 I, a radioisotope which can be detected and quantified with a gamma counter.
  • plaque reduction assay or "PRA,” as used herein describes a standard method used to determine efficacy of anti-viral drugs by enumerating a decrease in plaque formation in a cell monolayer exposed to a drug.
  • a “plaque” is a defined area of “CPE.” It is usually the result of infection of the cell monolayer with a single infectious virus which then replicates and spreads to adjacent cells of the monolayer.
  • a plaque may also be referred to as a "focus of viral infection.”
  • transmissive describes the sequence of interactive events between a virus and its putative host cell. The process begins with viral adsorption to the host cell surface and ends with release of infectious virions. A cell is "permissive” if it readily permits the spread of virus to other cells. Many methods are available for the determination of the permissiveness of a given cell line, including but not limited to, plaque reduction assays, comparisons of the production and/or quantitation of viral proteins based on results obtained from gel electrophoresis, relative comparisons using hybridization analysis to analyze DNA or RNA content, etc.
  • permissive as used herein describes the extent that a permissive or non-permissive host cell can adsorb and be penetrated by a virus.
  • a cell line may be susceptible without being permissive in that it can be penetrated but not release virions.
  • a permissive cell line however must be susceptible.
  • seed on describes the act of transferring an aqueous solution of suspended ceils into a vessel containing cells adhered to a surface, after which the vessel is stored for a sufficient period of time to allow the suspended cells or “seeds” to settle out by gravity and attach in a relatively uniform manner to the adhered cells and become integrated into the final cell monolayer as a mixture.
  • a “mixed cell monolayer,” results from the “seed on” process.
  • seed in describes the mixing of two or more aqueous solutions of suspended tissue culture cells, each cell suspension having different cellular properties, and transfer of such mixture of ceils into a vessel which is stored for a sufficient period of time to allow the suspended cells to settle out by gravity and attach in a relatively uniform manner such that the distribution of any single cell type is indicative of the relative ratio of the cells in the original mixture.
  • starts refers to the reporter cells which represent a primary infection of virus.
  • the virus infects a reporter cell (a genetically engineered cell) and induces the expression of the reporter gene.
  • a reporter cell can be nonpermissive (i.e. permissiveness of the reporter cells is not required) and still produce starts.
  • chimeric refers to any nucleic and/or amino acid sequence containing portions from two or more different species.
  • a protein may be chimeric if the primary amino acid sequence contains portions from two or more different species (i.e., for example, an hTSH/rLH-R or RMc4).
  • a protein may also be chimeric if the primary amino acids sequence contains portions from two or more different proteins, whether from the same species or different species.
  • a protein may also be chimeric if the quaternary amino acid structure contains proteins from two or more different species.
  • a nucleic acid may be chimeric if the primary nucleotide sequence contains portions from two or more different species.
  • a nucleic acid may also be chimeric if the primary nucleotide sequence contains portions from two or more different proteins, whether from the same species or different species.
  • glucocorticoid refers to any compound any corticosteroid that increases gluconeogenesis, raising the concentration of liver glycogen and blood glucose; the group includes, but is not limited to, dexamethasone, prednisone, hydrocortisone, fluticasone, Cortisol, cortisone, or corticosterone.
  • the present invention provides methods and compositions useful in the diagnosis of autoimmune diseases.
  • the present invention provides methods and compositions for use in the diagnosis and management of Graves' disease.
  • one composition comprises a chimeric thyroid stimulating hormone receptor having improved sensitivity and specificty for circulating thyroid stimulating immunoglobulin. Assays using such chimeric receptors can be optimized in the presence of a glucocorticoid.
  • the present invention provides methods and compositions for monitoring the immune status and responses of individuals.
  • the present invention finds use in montoring the immune responses of vaccine recipients.
  • the present invention further provides methods and compositions for accelerating and enhancing the attachment of viruses to cell surface receptors, providing increased sensitivity in assays to detect and quantitate viruses in samples.
  • the clinical picture of Graves' disease in young adults is very easily recognized.
  • the patients are more commonly female than male, and report symptoms including, but not limited to, sweating, palpitations, nervousness, irritability, insomnia, tremor, frequent stools, and weight loss in spite of a good appetite.
  • Physical examination usually shows mild proptosis, stare, lid lag, a smooth, diffuse, non-tender goiter, tachycardia (especially after exercise) with loud heart sounds, and often a systolic murmur or left sternal border scratch, tremor, onycholysis, and palmar erythema; often, a Sonar erythema; often, a Sonar erythema; often, a Sonar erythema; often, a Sonar erythema; often, a Sonar erythema; often, a Sonar erythema; often, a Sonar erythema; often, a Sonar erythem
  • Graves' disease is readily recognized, and can be confirmed with laboratory tests (See, Federman, Thyroid, in Dale and Federman (eds.), Scientific American Medicine, 3:1-6, Scientific American, New York, NY, (1997).
  • the signs and symptoms described above can be troublesome, other symptoms of the disease can be more dangerous.
  • One of the most disturbing symptoms is severe exophthalmos, accompanied by ophthalmoplegia, follicular conjunctivitis, chemosis, and loss of vision.
  • Additional symptoms include, but are not limited to, dermopathy, pretibial myxedema, clubbing, and in the most severe cases, acropachy. These signs and symptoms are indicative of a representative autoimmune etiology of Graves' disease.
  • RAIU radioactive-iodine uptake
  • thyroid-stimulating autoantibodies e.g., cAMP generation
  • TSH radiolabeled thyroid stimulating hormone
  • JP09 cells Choinese hamster ovary cells transfected with a functional human TSH receptor
  • other cell lines which stably express the human TSH receptor have greatly improved the assay systems available for the detection of Graves' disease autoantibodies.
  • These cells have a TSH receptor that is comparable to that of native thyrocytes and possess a functional signal transduction system involving G-protein coupling, activation of adenylate cyclase and c AMP generation in response to TSH and to thyroid-stimulating antibodies (TSAb) (See e.g., Michelangeli et al., supra).
  • TSAb thyroid-stimulating antibodies
  • FRTL-5 cells have been reported to be superior to FRTL-5 cells as they provide similar diagnostic information, but are more sensitive, grow faster, have less fastidious growth requirements, and respond to unextracted sera, in comparison with FRTL-5 cells (Michelangeli et al., supra; see also, Kakinuma et al., J. Clin. Endocrinol. Metabol, 82:212902134 (1997)).
  • these methods are more rapid and reproducible, and perhaps more specific for detection of human autoantibodies directed against the human receptor.
  • the assays are easier and less cumbersome to perform than those using the FRTL-5 cell line (See e.g., Vitti etal., J. Clin. Endocrinol.
  • Graves' disease is a thyroid disorder caused by an antibody-mediated autoimmune reaction.
  • the autoantibodies recognizing the TSHR are heterogeneous, including mainly thyroid stimulating antibodies (TS Abs) and thyroid blocking antibodies (TBAbs.)
  • TSAbs act as a TSH agonist causing hyperthyroidism while the TBAbs function as a TSH antagonist causing hypothyroidism.
  • TBAb binding can "neutralize" the stimulating effect of TSAb.
  • the TSAb binds to the TSHR, it induces the cAMP signaling pathway, TBAb does not have this effect.
  • the Kronus ® Radio Receptor Assay (RRA) kit is used for determination of TRAbs and detects both TSAbs and TBAbs but cannot distinguish between the two.
  • Diagnostic Hybrids Inc. DHI
  • DHI previously developed a Graves' diagnostic CHO-Luc cell line that detects the TRAbs in patient serum. This cell line co-expresses the wildtype TSH receptor gene and a firefly luciferase gene which is driven by the human glycoprotein alpha subunit promoter.
  • This wild type TSHR has epitopes that bind TSAbs and TBAbs.
  • Thyroid-stimulating autoantibodies directed against the thyroid stimulating hormone (TSH) receptor are capable of stimulating thyroid adenylyl cyclase, the enzyme responsible for producing cyclic-adenosine monophosphate (cAMP).
  • Thyroid-stimulating autoantibodies directed against the thyroid stimulating hormone (TSH) receptor are capable of stimulating thyroid adenylyl cyclase, the enzyme responsible for producing cyclic-adenosine monophosphate (cAMP).
  • Thyroid-stimulating autoantibodies directed against the thyroid stimulating hormone (TSH) receptor are capable of stimulating thyroid adenylyl cyclase, the enzyme responsible for producing cyclic-adenosine monophosphate (cAMP).
  • FRTL- 5 rat thyroid cell line
  • This cell line available from Interthyroid Research Foundation (Baltimore, MD) expresses receptors that cross-react with human TSAbs.
  • TSAbs i.e., for example, upon exposure of the cells to serum from a Graves' patient containing these antibodies
  • FRTL-5 cells are stimulated to produce cAMP.
  • This cAMP is then measured in a portion of the lysed cells or the medium bathing the cells using a radioimmunoassay method.
  • the FRTL-5 cells formed the basis for the first successful bioassay for the autoantibodies that are pathognomonic of Graves' disease.
  • U.S. Patent No. 4,609,622 herein incorporated by reference).
  • a typical assay using FRTL-5 cells performed as described by Vitti et al. involves seeding FRTL-5 cells in 96- well plates (30,000 cells/well) in a special complete medium containing 6 hormones (i.e., for example, a 6H medium) in addition to the normal growth constituents used in cell culture medium.
  • a 6H medium a special complete medium containing 6 hormones
  • the medium is changed to a "Starvation Medium," which is deficient in TSH (thereby resulting in a 5H medium), wherein TSH is one of the 6 hormones in the 6H medium.
  • the cells are then maintained for 4-5 days in the incubator with a medium change every 2-3 days. During this time the cells do not grow or multiply. Subsequently, the cells may be used in a diagnostic assay.
  • luciferase is measured simply by removing the medium from the cells, adding a lysis buffer, allowing 20-30 minutes for lysis to occur, removing a sample of the lysate, adding luciferase substrate and measuring light output over a 15 second interval using a luminometer.
  • this method provides equivocal results and required further improvement.
  • the present invention contemplates methods that incorporate the advantages of a CHO-Rluc protocol, while providing additional advantages in terms of reliability and reproducibility.
  • Considerable development effort was dedicated to the development of methods of the present invention, including those that allow the use of CHO-Rluc cells in luminometric assays using TSH and immunoglobulins from untreated Graves' disease patients.
  • the standard protocol originally used involved planting the CHO-Rluc cells from a frozen stock, so as to seed at a concentration that produced confluent monolayers after 18-24 hours of incubation. Initially, the Growth Medium was removed and Stimulation Medium was added to the monolayers, to which a series of TSH standards (e.g., 0, 10, 100, 1000 ⁇ lU TSH/ml), and patient IgG samples were added. As this approach yielded poor results, an overnight Starvation or conditioning period was tested. A Starvation period resulted in improved results with lower background values and appeared to produce good values for the TSH standards and the test patient samples. An additional experimental option was also tested in which polyethylene glycol (PEG) was used to enhance antigen and antibody binding.
  • PEG polyethylene glycol
  • Starvation with standard HBSS resulted in RLU/sec values of (21,671) for the 0 ⁇ U/ml TSH control, 1,336 for the 10 ⁇ lU TSH/ml sample, 82,466 for the 1000 ⁇ lU TSH/ml sample, and .39,082 for IgG sample #13.
  • Starvation with standard HBSS and 6% PEG in the Stimulation Medium resulted in RLU/sec values of (32,562) for the 0 ⁇ lU/ml TSH control, 5,980 for the 10 ⁇ lU TSH/ml sample, 207,831 for the 1000 ⁇ lU 5 TSH/ml sample, and 174,461 for IgG sample #13.
  • the present invention contemplate recombinant cell lines (i.e., for example, CHO and RD) that express a TSH/LH/TSH chimeric receptor (i.e., for example, RMc4) in combination with a firefly luciferase gene.
  • the expression is driven by a human glycoprotein alpha subunit promoter.
  • a chimeric receptor comprises at least one genetic modification such that only a TSAb binding region is expressed. It is believed that the recombinant cell lines have increased specificity when compared to either the CHO-Luc cells or KRONUS ® assay.
  • the present invention also provides methods and compositions for the monitoring of immune response development.
  • the present invention provides methods and compositions suitable for monitoring the response of individuals to vaccination.
  • a pre-immune serum i.e., serum collected prior to administration of vaccine
  • Such serum would also be collected shortly following vaccination (e.g., 1-2 weeks after vaccination), as well as periodically in the months following vaccination.
  • the serum samples are then tested for the presence and quantity of neutralizing antibodies.
  • diagnostic assays are conducted to monitor the response to viral antigens.
  • cells such as ELVISTM (Diagnostic Hybrids, Athens, OH) are used in combination with a polyethylene glycol (PEG) solution of the present invention.
  • PEG polyethylene glycol
  • PEG enhances the antigen-antibody reaction, thereby resulting in higher reactivity.
  • the present invention contemplates using genetically engineered Chinese Hamster Ovary (CHO) and/or human Rhabdomyosarcoma cells (RD) for diagnosing Graves' disease and/or monitoring Graves' disease therapy.
  • CHO Chinese Hamster Ovary
  • RD human Rhabdomyosarcoma cells
  • Clinical laboratories currently utilize various cells and reaction buffer for the detection and measurement of stimulating autoantibodies specific to Graves' disease in patient sera for identifying patients suffering from this disease and monitoring their therapy.
  • cells comprising genetically modified CHO cells containing wild type human Thyroid Stimulating Hormone Receptor (TSHR) and the CRE-Luc reporter system are utilized by numerous laboratories. These cells, however, need one day for growth and one day for starvation which puts a time constraint on test results availability.
  • TSHR human Thyroid Stimulating Hormone Receptor
  • CRE-Luc reporter system are utilized by numerous laboratories. These cells, however, need one day for growth and one day for starvation which puts a time constraint on test results availability.
  • the patient's serum specimens are incubated with the cells and reaction buffer in order to detect the presence of the Graves' autoantibodies.
  • the present invention contemplates methods that do not require these multi-day assay procedures, hi one embodiment, these shorter methods do not have a Starvation period incubation.
  • TSI thyroid stimulating immunoglobulin
  • the cell line further comprises a chimeric receptor.
  • the chimeric receptor comprises a human Thyroid Stimulating Hormone Receptor (TSHR) and a rat Luteinizing Hormone (LH) (i.e., for example, a RMc4 receptor).
  • TSHR human Thyroid Stimulating Hormone Receptor
  • LH Luteinizing Hormone
  • a chimeric TSH receptor provides improved binding specificity for TSI such that a Starvation period in the diagnostic assay is not required.
  • the present invention contemplates a method for expressing the Mc4 chimeric receptor in the CHO cells and/or RD cells (or other mammalian cells).
  • the method further comprises using CRE-Luc as a reporter gene to detect TSI.
  • the chimeric receptor provides greater specificity than a wild-type receptor by preferentially binding to stimulating autoantibodies (i.e., as opposed to blocking autoantibodies).
  • the chimeric receptor provides greater sensitivity than a wild-type receptor by preferentially binding to stimulating autoantibodies (i.e., as opposed to blocking autoantibodies).
  • the cell culture further comprises PEG.
  • the cell lines do not need to be "starved” overnight, a requirement for currently used cell lines in order to maximize the signal resulting from TSI binding. This change reduces the turn-around time from a current 3 day assay to a 2 day assay, which is very advantageous to the laboratory, the physician, and the patient.
  • the assay is designed to measure stimulating antibodies, whereas the wild type TSH-R is responsive to both stimulating and blocking antibodies whereas this Mc4 chimeric receptor is responsive only to stimulating antibodies, thereby providing greater specificity for what is being measured.
  • the present invention contemplates novel diagnostic cell lines that detect thyroid stimulating hormone receptor (TSH-R) autoantibody (i.e., for example, thyroid stimulating immunoglobulin; TSI) with high detection sensitivity and specificity.
  • TSH-R thyroid stimulating hormone receptor
  • the cell line comprises a recombinant Chinese Hamster Ovary cell (i.e., for example, a CHO-Kl cell).
  • the cell line comprises a Human Rhabdomyosarcoma (RD) cell.
  • the present invention contemplates a vector comprising a nucleic acid sequence encoding a hTSH/rLH-R fusion protein (i.e., for example, RMc4) linked to a firefly luciferase reporter gene and in operable combination with a glycoprotein hormone alpha subunit promoter.
  • a cell line is transfected with the vector.
  • the transfected cell line expresses a human TSH-R/rat Luteinizing hormone (LH) chimeric receptor (hTSH/rLH-R), under conditions such that the luciferase reporter signal is detected.
  • LH Luteinizing hormone
  • the heterogeniety of anti-TSH receptor antibodies was addressed by comparing binding of: i) TSH-binding inhibitory immunoglobulin; ii) thyroid-stimulating antibody; and iii) thyroid blocking antibody using a chimeric human TSH receptor wherein amino acid residues 90-165 of the human TSH receptor were substituted by equivalent amino acid residues from the lutenizing hormone chorionic gonadotropin receptor.
  • the binding data suggest that there might be two different types of thyroid-stimulating antibodies, three different types of TSH- binding inhibitory immunoglobulins, and one nonfunctional antibody.
  • Chimeric TSH receptors have been reported to detect and characterize various types of circulating antibodies suspected of having a relationship with Graves' disease. Such antibodies are believed to include, but are not limited to, stimulating autoantibodies that can activate TSH-R and blocking autoantibodies that can block TSH-R binding by either TSH or stimulating autoantibodies.
  • stimulating autoantibodies that can activate TSH-R
  • blocking autoantibodies that can block TSH-R binding by either TSH or stimulating autoantibodies.
  • chimeras of human TSH-R (hTSH-R) and lutenizing hormone human chorionic gonadotropin receptor (LH-hCG-R) included an RMc4 chimera having amino acids 261-370 of the hTSH-R substituted with equivalent residues from a human LH/CG-R.
  • the interactions between TSH stimulating and blocking autoantibodies was addressed by using two types of TSH-R chimera constructs.
  • the first chimera is designated Mc2 having human TSH-R amino acid residues 90-165 substituted by equivalent residues from rat lutenizing hormone chorionic gonadotropin receptor.
  • the second chimera is designated Mc 1+2 having human TSH-R amino acid residues 8-165 substituted by equivalent residues from rat lutenizing hormone chorionic gonadotropin receptor.
  • the sequence that drives the expression of the luciferase reporter is a 236 nucleotide glycoprotein alpha subunit promoter, which contains a cyclic AMP (cAMP) regulatory element (CRE) and was cloned by PCR.
  • the nucleotide sequence of the cloned promoter was determined by DNA sequencing and was confirmed by sequence comparison with Gene bank sequence AF401991. An alignment of the cloned promoter with a GPH promoter amplified by PCR from HEK cells indicate that the two sequences are identical.
  • detecting sensitivity between the CHO-Rluc, CHO-RMc41uc and RD- RMc41uc cell lines induced with a serially diluted TSI positive serum was compared.
  • a TSI positive serum was serially diluted and incubated on the different cell lines for three hours.
  • the RD-RMc41uc and CHO-Rluc cell lines showed linear responses of the ratio of S/N in the serum dilution range between 1 :2 and 1 :8.
  • the slope of the dose response (value) and hence, the detection sensitivity, for RD- RMc41uc was much higher than that of CHO-Rluc cell line.
  • CHO-RMc41uc did not show a linear response of the ratio of S/N at no or low serum dilutions.
  • Figure 1 IB. CHO-RMc41uc cells, however, did show a linear dose response of the ratio of S/N from the serum dilution ranging from 1 :32 to 1 : 128.
  • Figure 11C. Note that the slope of the dose response (value) was even higher than that of RD-RMc41uc cell line.
  • the CHO-RMc41uc cell line was also compared to the CHO-Rluc cell line for TSH sensitivity.
  • the S/N ratio was derived from the luciferase assay using CHO-Rluc, CHO-RMc41uc and RD-RMc41uc cell lines induced with recombinant human TSH.
  • CHO-RMc41uc and CHO-Rluc cell lines were also tested for their specificity using other anterior pituitary hormones including human luteinizing hormone, (hLH,) human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG), all of which share a common alpha subunit. Neither cell line showed any cross activity with the tested hormones.
  • Table 1. Table 1. Specificity of CHO-Rluc and CHO-RMc41uc to human TSH and other hormones.
  • CHO-RMc41uc and CHO-Rluc cell lines were used to screen normal human sera to determine the distribution of the ratio of S/N derived from a luciferase assay. Comparisons of distribution of the S/N ratios derived from luciferase assays on CHO- Rluc and CHO-RMc41uc cell lines induced with sera from 108 normal people were performed. All serum samples were tested in both CHO-RMc41uc and CHO-Rluc cell lines. A known normal serum was used as a reference for calculating S/N ratios. The distribution of CHO-RMc41uc cell line revealed a pattern very similar to that of the CHO- Rluc cell line. The mean of the CHO-Rluc cell was 1 and the CH0-RMc41uc was 0.88. The standard deviation of CHO-Rluc was 0.23 and CHO-Luc was 0.21. Figure 13.
  • the responses of the CHO-RMc41uc, RD-RMc41uc and CHO-Rluc cell lines to clinical patient serum samples were compared.
  • the ratio of S/N derived from the luciferase assay on CHO-Rluc, CHO-RMc41uc and RD-RMc4 luc cell lines induced with 12 clinical serum samples.
  • Each of the 12 serum samples was tested in the CHO-
  • the CHO-RMc41uc cell line provides definite advantages in sensitivity and specificity over the currently used CHO-Rluc cell lines.
  • the CHO-RMc41uc cells upon stimulation by Graves' Disease antibodies, provide increased luciferase responses as measured by the Relative Light Units (RLU) output.
  • RLU Relative Light Units
  • This improvement allows the elimination of the one day starvation step from the protocol.
  • the standard procedure used for the CHO-Rluc cell line includes a starvation period.
  • the protocol for the Starvation format is to plant the cells for Growth on day 1, Starve on day 2 and Stimulate and measure RLU on day 3.
  • the RLU values for Graves' Positive and Negative sera as obtained using the respective protocols for starved CHO-Rluc and CHO-RMc41uc cells, and non-starved CHO-RMc41uc cells.
  • the intended effect of starvation on CHO-RMc41uc cells was to decrease the background level of luciferase activity in the cells, thereby raising the ratio of RLU between Graves' Positive and Graves' Negative sera.
  • the average ratio for Starved CHO-Rluc is 3.25x
  • for non-Starved CH0-RMc41uc is 7.29X
  • Starved CHO-RMc41uc cells is 11.5X.
  • the non-starved CHO-RMc41uc cells provide a greater than 2-fold increase in luminosity (i.e., and therefore sensitivity) over the starved CHO-Rluc cells.
  • This provides distinct advantages of a protocol lasting one day shorter and providing more accurate and sensitive results.
  • the Serum:Reference Ratios (SRRs) compare the RLU values as percentages and further confirm that the CHO-RMc41uc cells (whether starved or non-starved) provide an improved separation between the RLUs between Normal and Graves' Disease sera.
  • the Cutoff values presented are approximate and are indicative of Graves' Disease when the assayed value is > to the cutoff value of the particular protocol/cell line.
  • RMc41uc cells underwent Starvation periods and then were incubated with various concentrations of dexamethasone (DEX). The data clearly indicate that dexamethasone significantly improved the RLU intensity over and above that provided by a Starvation period alone. Table 9.
  • dexamethasone reduces the Reference serum RLU readings while at the same time greatly increasing Patient #18 RLU readings. Overall, the presence of dexamethasone results in about an 80% increase in S/B ratios for the Patient #18 serum samples.
  • CHO-rMc41uc cells with dexamethasone provides a more sensitive (and therefore more accurate) detection of circulating TSI's as compared to non-starved CHO-RMc41uc cells without dexamethasone.
  • CHO-rMc41uc cells with dexamethasone showed a 365% increase in luminescence (as relative to the Reference) while CHO-rMc4 cells without dexamethasone showed a 189% increase in luminescence.
  • dexamethasone provides a better assay system in terms of sensitivity due to a stronger luminescent signal strength.
  • the present invention contemplates a diagnostic assay that is more rapid and accurate than any previously disclosed TSI antibody assay requiring a Starvation medium peroid.
  • this effect was not limited to dexamethasone but can be expected from most, if not all, glucocorticoids.
  • the data presented herein show that other glucocorticoids also improve the sensitivity of the CHO-RMc41uc assay to provide equivalent sensitivity in comparison with substitution for a Starvation medium period. Nonetheless, the presence of a glucocorticoid provides the advantage that the assay can be performed in two days, rather than three days.
  • glucocorticoids where compared to dexamethasone (40 ⁇ M) on the basis of Relative Light Units (RLUs) and Serum Reference Ratios expressed in percentages (SRRs%).
  • RLUs Relative Light Units
  • SRRs% Serum Reference Ratios expressed in percentages
  • the data was collected in accordance with the protocol outlined in Example 17.
  • the data was calculated as the difference ( ⁇ ) in RLU values or SRR% values for each glucocorticoid (GC) concentration by subtracting the RLU value or SRR% value for the Normal control at that concentration from the RLU value or SRR% value for the Positive control at that concentration, respectively.
  • kits for performing Graves' disease diagnostic assays using chimeric TSH receptors preferably include one or more containers containing a cell line-based diagnostic method of this invention.
  • the containers may contain a glucocorticoid including, but not limited to, dexamethasone, cortisone, hydrocortisone, prednisone, or fluticasone.
  • the kits contain all of the components necessary or sufficient for performing a Grave's disease diagnostic assay to detect circulating TSH autoantibodies in patient sera, including all controls, directions for performing assays, and any software for analysis and presentation of results.
  • kits contain vectors encoding chimeric TSH receptors capable of transfecting cell lines.
  • the kits comprise all materials necessary or sufficient to perform diagnostic assays in a single reaction and provide diagnostic, prognostic, or predictive information (e.g., to a researcher or a clinician).
  • a kit might contain a cell line comprising a chimeric TSH receptor and a luciferase reporter system.
  • the kits comprise one or more of a vector comprising a first nucleic acid sequence for an Mc4 chimeric TSH receptor, a second nucleic acid sequence for a luciferin/luciferase reporter system, and a third nucleic acid sequence for a promoter.
  • kits may also optionally include appropriate systems (e.g. opaque containers) or stabilizers (e.g. antioxidants) to prevent degradation of the reagents by light or other adverse conditions.
  • appropriate systems e.g. opaque containers
  • stabilizers e.g. antioxidants
  • Each solution or composition may be contained in a vial or bottle and all vials held in close confinement in a box for commercial sale.
  • kits may optionally include instructional materials containing directions (i.e., protocols) providing for the use of the reagents in the diagnosis, detection, and/or treatment of Graves' disease.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • Such media may include addresses to internet sites that provide such instructional materials.
  • GM or PM Growth Medium or Planting Medium
  • SM Startvation Medium
  • HBSS Hank's Balanced Salt Solution
  • EMEM Eagle's Minimum Essential Medium
  • FBS or FCS fetal bovine serum or fetal calf serum
  • DMSO dimethyl sulfoxide
  • CHO Choinese hamster ovary cells
  • CHO-R CHO cells transfected with the human TSH receptor
  • CHO-Rluc CHO-R cells transfected with the cre-luciferase reporter gene complex
  • Biochemical U.S. Biochemical Corp., Cleveland, OH
  • Fisher Fisher
  • Sigma Sigma
  • ATCC American Type Culture Collection, Rockville, Maryland
  • LTI Life Technologies, Rockville, MD
  • Promega Promega Corp., Madison, WI.
  • All solutions used in these methods were sterile (with the exception of TSH, controls, patient specimens) and treated aseptically. All manipulations were conducted in a biosafety cabinet under aseptic conditions.
  • Cell culture media e.g., Ham's F- 12, EMEM, etc.
  • additive reagents such as non-essential amino acids were obtained from Sigma.
  • Freezer vials of cells should not be allowed to warm from their -80 0 C (or lower) storage temperature until immediately prior to thawing and use in the methods of the present invention, as cycling of the temperature may result in viability losses. Because it contains dithiothreitol, which is unstable at room temperatures, the 5x cell lysis solution should be removed from its -20 0 C storage temperature only long enough to remove the required volume for preparation of the Ix solution. As it also contains dithiothreitol, reconstituted luciferase substrate solution should be kept frozen at -20°C until just prior to use, at which time it may be removed and placed in a 25-37°C water bath to thaw and reach room temperature.
  • the liquid when removing liquid from wells (e.g., microtiter plates, etc.), the liquid may be dumped from the wells into a receptacle in a biosafety hood.
  • the residual liquid can be drained and removed by placing the plate upside down on a sterile, absorbent wipe.
  • the liquid may be removed by aspiration using a fine tip on the aspirator. If aspiration is used, the plate is held at a steep angle so that the liquid does not overflow the wells, and the aspirator tip is directed down the side of the well almost to the bottom to remove the liquid and only leave minimal residue.
  • care must be exercised in order to prevent disturbance of the cell monolayer, as the cells can be easily removed by the aspirator.
  • the cells be distributed evenly within the wells.
  • the transfer of cell suspensions into wells should be performed in a vibration- free biosafety hood. After all of the wells in a plate have received cells, the plate is covered and carefully placed on a solid, vibration- free surface, for 30 minutes, to allow the cells to attach undisturbed, to the bottom of the wells. This helps ensure that an even distribution of cells is present in each, of the wells.
  • CHO-Rluc cells were prepared from W-25 CHO-R cells for use in the testing methods to detect TSI in Graves' disease patients. Pools of puromycin- resistant cells were obtained and tested for light output in response to bovine TSH. Clones with the highest light output were selected for use in the experiments described below.
  • CHO-Rluc cells were grown in cell culture flasks (e.g., T-225 flasks) in growth medium containing Ham's F-12 medium, 10% FBS (heated at 56°C for 30 minutes to inactivate complement), 2 mM glutamine, and Ix non-essential amino acids. The flasks were incubated at 35-37°C, in a humidified atmosphere, containing 5% carbon dioxide.
  • the medium from each flask was aspirated, and the cell monolayers were washed with HBSS without Ca and Mg 4+ . Then, 7 ml of a 0.25% trypsin/1 mM EDTA solution were added to each flask, and allowed to react with the monolayers for approximately 5-10 minutes at room temperature, in order to detach and disperse the cells in a nearly unicellular suspension. The cell suspensions were then centrifuged for approximately 5 minutes at 300-400 x g.
  • the supernatants were then removed and the pelleted cells resuspended in 8 ml of a medium prepared by mixing 4 ml EMEM containing Ix HBSS and 20% FBS with 4 ml of cryoprotective medium (EMEM containing Ix HBSS and 15% DMSO).
  • each cell suspension was then used to determine the number of cells present in the suspension. This determination can be accomplished using any method known in the art, including but not limited to methods using a hemocytometer to determine the cell count. Thus, it is contemplated that any method can be used to determine the cell count in the suspensions. Based on the number of cells in the suspension, the cells were aliquoted by volume to approximately 2 x 10 6 cells into standard freezer vials. The cells were then stored frozen at -90°C for short-term storage. For long-term storage, the cells were stored in liquid nitrogen (about -200°C). EXAMPLE 2
  • CHO-Rluc cells prepared as described in Example 1 were used in assays for diagnosis of Graves' disease.
  • 24 wells in a 96-well microtiter plate were first treated by adding 50-100 ⁇ l 0.1% gelatin solution (Sigma) to enhance attachment of the cells to the bottom of the 24 wells chosen for the test.
  • the gelatin solution was removed from each of the wells by aspiration. It was noted that the gelatin can remain on the wells for longer than one minute.
  • the gelatin serves to coat the wells with collagen, so that the cells attach more quickly to the wells and reach confluence more rapidly. However, cells can be planted and grown to confluence without gelatin and still perform well.
  • a freezer vial of CHO-Rluc cells produced as described in Example 1 was rapidly thawed in a 37°C water bath to provide approximately 0.4 ml cell suspension, which was well-mixed using a pipette.
  • the cells were then added to 2.5 ml GM (also referred to as "Planting Medium"), thoroughly mixed by vortexing for 1-2 seconds, and 100 ⁇ l aliquots of the cell suspension were added to each well, and the plates were covered. It is preferable to produce an even distribution of cells in each well.
  • the microtiter plate should be placed in a vibration- free hood for cell planting and attachment of cells to the walls of the microtiter plate.
  • the planted cells were then incubated at 35-37°C, in a humidified atmosphere, containing 5% CO 2 , for approximately 20-24 hours, to allow the cells to form a nearly or completely confluent monolayer.
  • the GM was then aspirated from each well as completely as possible, being careful not to disturb the monolayers (i.e., confluent monolayers remain in the wells).
  • the monolayers were rinsed with approximately 100 ⁇ l Starvation Medium (HBSS containing Ca +" * " (0.14 g/L) and Mg +4" (0.048 g/L) per well.
  • the Starvation Medium was aspirated and a fresh 100 ⁇ l of Starvation Medium was then added to each well. It is important that these steps be conducted sufficiently rapidly that the cell monolayers do not dry.
  • the plates were then incubated overnight in a 35-37°C, 5% CO 2 , humidified incubator. Following incubation, the Starvation Medium was aspirated from the wells, using care to avoid disturbing the monolayers. Then, approximately 100 ⁇ l Stimulation Medium were added to each monolayer, again working quickly so that the monolayers did not dry.
  • TSH standards and IgG samples were diluted with diluent (i.e., HBSS-NaCl + 222 mM sucrose).
  • the TSH standards were tested at concentrations of 0, 10, 100, 1000, and 5000 ⁇ lU.
  • Patient samples were diluted to a concentration of 10 mg protein/ml for use in the assay.
  • Stimulation Medium is viscous, thorough mixing of the suspensions was important. Adequacy of the mixing was ascertained by microscopic examination of the monolayers. The plates were incubated for 4 hours at 35-37°C in a 5% CO 2 , humidified incubator.
  • the medium was carefully aspirated from each well and 150 ⁇ l lysis solution (Promega) was added to each well.
  • the lysis solution contained 25 mM Tris-phosphate, pH 7.8, 2 mM diaminocyclohexane tetraacetic acid (CDTA), 2 mM dithiothreitol (DTT), 10% glycerol, and 1 % Triton X-100.
  • CDTA diaminocyclohexane tetraacetic acid
  • DTT dithiothreitol
  • glycerol glycerol
  • Triton X-100 Triton X-100
  • the average net value for the zero ⁇ U/ml TSI standard was 68,01 1 RLU/sec, while the result for the sample containing 10 ⁇ lU/ ⁇ l was 4031 RLU/sec, the sample containing 1000 ⁇ lU was 222,801 RLU/sec, one Graves' IgG test sample was 384 RLUTsec (sample #1), and another Graves' IgG test sample was -3012 RLU/sec (sample #9).
  • the Graves' IgG sample #1 and sample #9 were previously assayed using standard FRTL-5 cells and a cAMP RIA assays.
  • the luciferase in the cell lysates was measured by adding 25 ⁇ l of lysate to a luminometer tube to which 50 ⁇ l of substrate solution (Promega) were added. The suspensions were mixed and then read in a luminometer with settings of a 5 second delay and a 10 second read, to determine the RLU for each sample.
  • the Starvation Medium was removed by aspiration, and 100 ⁇ l of the Stimulation Medium were added to each well.
  • This Stimulation Medium was HBSS-NaCl + 222 mM sucrose.
  • Table 2 rovides a comparison of the formulations of HBSS-NaCl + 222 mM sucrose and standard HBSS. Table 2.
  • This Stimulation Medium formulation is a formulation that is commonly used in the measurement of TSI in FRTL-5 and CHO-R cells.
  • a CHO GM is CHO Growth Medium containing 10% FBS.
  • b CHO Char is CHO Growth Medium with 10% charcoal-stripped calf serum.
  • c A Starvation Medium EXAMPLE 6 Use of PEG
  • PEG may be used in in vitro antigen/antibody reactions to assist or enhance the reaction rate
  • a trial was conducted in which PEG was incorporated into the Stimulation Medium.
  • this compound may decrease the off-rate or dissociation of the antigen/antibody complex
  • the use of PEG in the methods of the present invention was investigated.
  • the IgG samples were prepared from 10 mg/ml stocks, which were then tested undiluted, and serially diluted (3-fold dilutions) to 0.3333, 0.1111, 0.0371, 0.0123, and 0.0041 dilutions (i.e., to yield 3.333 mg/ml, which was then diluted 3-fold to yield 1.111 mg/ml, etc.).
  • the FRTL-5 value for IgG sample #6 was 2080, while the FRTL-5 value for IgG sample #11 was 4453, and for IgG sample #16, the value was 830.
  • the following Table 6 lists the results for each of these samples.
  • the correlation coefficients (r) were 0.857 for IgG sample #6, 0.858 for sample #11, and 0.995 for sample #16.
  • the Growth Medium was removed and the monolayers rinsed with 100 ⁇ l of Starvation Medium (normal HBSS with Ca ++ and Mg +4 ), and a final 100 ⁇ l were added to each monolayer before incubating overnight under the conditions described above. Following incubation, the Starvation Medium was removed and 100 ⁇ l of Stimulation Medium containing 6% PEG (i.e., as described above) were added to each monolayer. Then, 10 ⁇ l of each of the standards and samples were placed into the wells (in triplicate). While other volumes were tested (e.g., 25 ⁇ l, 50 ⁇ l, and 75 ⁇ l), the values obtained were substantially equivalent to those obtained with 10 ⁇ l volumes. Thus, the smaller volume was used in order to conserve the samples and reagents, and to minimize the concentration of potentially interfering substances present in some serum samples.
  • the well contents were mixed and the monolayers incubated as described above for 4 hours (i.e., a stimulation step).
  • the medium was removed from each well, and 150 ⁇ l of lysis solution (as described above) were added to each well.
  • the monolayers were allowed to stand at room temperature for 30 minutes for lysis to occur.
  • 25 ⁇ l of each lysate were added to individual luminometer tubes.
  • Fifty microliters of luciferase substrate (as described above) were added to each tube, the contents mixed, and the tubes immediately read in a luminometer with settings of 5 seconds delay and a 10 second read time.
  • Figure 2 provides a comparison of CHO-Rluc luciferase results with the FRTL-5 cAMP results. This Figure indicates that the correlation between these methods is quite good.
  • Figure 3 provides a comparison of CHO-Rluc luciferase results with CHO-R cAMP results.
  • the CHO-R CAMP cut-off value was 173. Values below this cutoff were as follows (CHOluc RLU/sec): 110 (219,913), 113 (14,434), 116 (25,373), 152 (84,493), 156 (7576), and 161 (61,321). As indicated in this Figure, the range of CHO-R cAMP results is relatively narrow, as compared with the CHO-Rluc values.
  • this linearity and sensitivity of response to bTSH will prove useful in the detection of blocking antibodies to the TSH receptor (e.g., those autoantibodies in patents with atrophic thyroiditis and Hashimoto's thyroiditis which block the TSH receptor, thereby preventing thyroid hormone production and release resulting in hypothyroidism).
  • This Figure also provides at least a partial explanation of why the CHO-R cell line is not as sensitive to TSI from Graves' disease patients sera as the FRTL-5 cell line. In these results, the correlation coefficient (r) was 0.9925. The three S.D. (standard deviations) sensitivity was 1.3 ⁇ lU TSH/ml.
  • this Example describes the monitoring of a subject's immune response to herpes simplex (HSV) vaccine.
  • a serum sample i.e., preimmune serum
  • Serum samples are also collected at periodic intervals following administration of the vaccine (e.g., 1-2 weeks, 1 month, 2 months post-vaccination, etc.).
  • the sera are thawed as necessary, and used in an assay to determine the presence and quantity (i.e., titer) of neutralizing antibodies.
  • Sera are serially diluted and mixed with known quantities of HSV. These samples are diluted in dilutent comprising Eagle's MEM with HBSS containing 2 mM glutamine, 2% FBS, and PEG (e.g., 6% PEG 8000).
  • diluents will find use in the present method, including diluents containing different concentrations and types of PEG, as appropriate for the virus and assay system used).
  • These samples are added to cell monolayers containing cells capable of producing an enzyme such as (3-galactosidase upon infection with HSV (e.g., ELVISTM cells, Diagnostic Hybrids).
  • an enzyme such as (3-galactosidase upon infection with HSV (e.g., ELVISTM cells, Diagnostic Hybrids).
  • the monolayers are lysed and the enzyme activity is measured using chromogenic or luminogenic methods.
  • a positive response to the vaccine is indicated by the lowest dilution of postvaccination serum which neutralizes HSV in the sample (i.e., as indicated by a low OD. or luminescence value, in comparison with the preimmune control).
  • the present invention provides numerous advances and advantages over the prior art, including the avoidance of radioactivity, in combination with the advantages of ease of use, reliability, sensitivity, specificity, cost-effectiveness, and reproducibility.
  • This example presents one embodiment of constructing a cell line comprising a chimeric TSH-R receptor for detecting Graves' disease autoantibodies.
  • a plasmid comprising a first nucleic acid sequences encoding a TSH-R chimeric receptor and a second nucleic acid sequence encoding a neomycin resistant gene was ligated to a luciferase gene and a glycoprotein hormone alpha subunit promoter.
  • Chromosomal DNA was isolated from human embryonic kidney cells using a QIAGEN RNA/DNA kit (QIAGEN Cat# 14123.) Glycoprotein alpha subunit promoter fragments were amplified by PCR using the isolated chromosomal DNA as the PCR template and the 2 pairs of oligo-nucleotide primers shown below: 5'PCR primer: 5'-GAGCTC ATG TGT ATG GCT CAA TAA AAT TAC GTA CAA AGT GAC AGC -3'
  • 3' PCR primer 5'- AGATCT TCG TCT TAT GAG TTC TCA GTA ACT GCA GTA TAA TGA AGT -3'.
  • a Sac I restriction site was added to the 5' end of the 5' PCR primer while a BgI II restriction site was added to the 5' end of the 3' PCR primer (both shown as underlined sequence).
  • BD Advantage 2 Polymerase Mix (BD Bioscience Palo Alto CA) was used and PCR reactions were performed in a thermal cycler (Eppendorf Mastercycler Personal, Germen.). Forty cycles were carried out at 94°C for 30 seconds to denature the DNA. Samples were then annealed to the primers in the thermalcycler at 63°C for 30 seconds, and the extension was induced at 68°C for 1 minute 30 seconds per cycle.
  • amplicons Two amplicons (1.2 kb and 0.6 kb) were cloned into the plasmid vector pcDNA2.1 (Invitrogen, Carlsbad, CA) and sequenced using the BigDye Terminator v3.0 Cycle Sequencing method on an ABI 377 automated sequencer (Davis Sequencing Inc.).
  • the human glycoprotein alpha subunit promoter was isolated from vector pcDNA2.1 by restriction cleavage with Sac I and BgI II. The resulting 316 bp fragment was then subcloned into the Sac I/Bgl II site of the pGL2 enhancer plasmid (Promega, Madison, WI) for construction of a plasmid named pGHP/Luc.
  • neomycin resistance gene for antibiotic selection (positive clone selection) was isolated from vector pMC 1 (Stratagene Cedar Creek, TX) with restriction enzymes of Xhol and HicII. The resulting fragment was then subcloned into the Xbal site of plasmid pMc4 that contains the TSHR/LH chimeric receptor driven by the SV40 promoter (from Dr. Leonard Kohn.) The final plasmid was named pMc4-neo.
  • pMc4-Bsd The antibiotic selection gene Blastocidin, isolated from vector pCMV/Bsd (Invitrogen, Carlsbad, CA) with restriction enzymes Xhol and Xbal, was subcloned into the Xbal site of plasmid pMc4 which contains the TSHR/LH chimeric receptor.
  • Tahara et al. "Immunoglobulins From Graves' Disease Patients Interact With Different Sites On TSH Receptor/LH/CG Receptor Chimeras Than Either TSH Or Immunoglobulins From Idiopathic Myxedema Patients" Biochem Biophys Res Comm
  • the human glycoprotein alpha subunit promoter with a firefly luciferase reporter gene, was isolated from vector pMc4/Luc followingrestriction cleavage by Smal and Accl. The isolated DNA fragment was then subcloned into the Pfol site of pMc4-neo plasmid. The final plasmid was named pMV4-GHP/Luc.
  • RD cells were transfected with the linearized pGPH/Luc (Seal) plasmid using HyFect (Denville Scientific, Metuchen, NJ.) according to the manufacturer's instructions. The cells were selected with 0.5 mg/ml of neomycin. The optimal clone from this transfection and selection was then transfected with the linearized plasmid pMc4-Bsd. After transfection, the cells were selected with both neomycin (0.5mg/ml) and blasticidin (5 ⁇ g/ml.) to produce the final RD recombinant cell line.
  • TSI-positive and normal serum All CHO and RD antibiotic resistant clones were tested with TSI-positive and normal serum to select the clone which can be used for the detection of TSI.
  • the TSI induction positive clone was subjected to the limiting dilution cloning to further select a single clone.
  • the final clones have the ability to diagnose Graves' disease and/or monitor the drug treatment of patients with Graves' disease with higher sensitivity than the current product on the market. These cell lines show good stability, having been passaged more than ten times, and continue to show very similar performance characteristics.
  • CHO cells from freezer vials were diluted and grown in growth media (Ham's F12 Medium with 10 (v/v) % fetal bovine serum and nine essential amino acids) for 16 hours at 37°C and 5% CO 2 . After 16 hours the media was removed and the CHO cells were rinsed and refed with 100 ⁇ l/ well "starvation" HBSS medium. The CHO cells were then incubated for 22-24 hours. Following incubation the media was removed and CHO cells were rinsed and refed with 100 ⁇ l/ well reaction buffer.
  • growth media Ham's F12 Medium with 10 (v/v) % fetal bovine serum and nine essential amino acids
  • the CHO cells were then induced with a 1 : 11 dilution of patient serum in reaction buffer containing BSA, PEG, sucrose, glucose, and salts (Diagnostic Hybrids Catalog number 40-300500;) for 4 hours at 37°C and 5% CO 2.
  • RD cells were grown in Eagles Minimal Essential Medium (EMEM) with 10(v/v) % fetal bovine serums at 37°C and 5% CO 2 for 16 -24 hours.
  • RD cells were then directly induced with patient serum in reaction buffer (Diagnostic Hybrids Catalog number 40- 300500) for 4 hours at 37°C and 5% CO 2. All publications and patents mentioned in the above specification are herein incorporated by reference.
  • CHO-Rluc (041307A) and CHO-RMc41uc (062707, P8, 3e6/ml) cells were each brought up in 5 ml CHO growth media. From this stock of cells enough was taken out and aliquoted into 6 tubes so that there would be 66,666 cells/well for CHO-Rluc assay and 50,000 cells/well for the CH0-RMc41uc assay for each dexamethasone concentration (100, 50, 40, 25, 12.5, and 0 ⁇ M). The dexamethasone stock was 500 ⁇ M in CHO-growth media. All dexamethasone was made fresh. These vials were spun down and the cells were brought up in the different concentrations of dexamethasone in CHO growth media.
  • CHO- Mc41uc cells (4e6 cells/plate) + Growth Medium containing 40 ⁇ M dexamethasone in CHO-Mc4 Reaction Buffer.
  • Plate 2 CH0-RMc41uc cells (4e6 cells/plate) + Growth Medium without 40 ⁇ M dexamethasone in CHO-Mc4 Reaction Buffer.
  • Plate 3 CHO- Rluc cells (4e6 cells/Plate) + Growth Medium without 40 ⁇ M dexamethasone in CHO- Rluc Reaction Buffer
  • Each plate underwent a 24 hour growth period, a 24 hour starvation period, a 4 hour induction period, and a 5 minute lysis period.
  • the luminosity for each sample was measured using a Veritas luminometer.
  • GCs were examined in this study: i) Prednisone (Sigma); ii) Hydrocortisone (Sigma); iii) Fluticasone Propionate (Sigma); and iv) Cortisone (Sigma).
  • a stock concentration (100 mM) of each GC was made in DMSO. 1:10 (10 mM) and 1 : 100 (1 mM) dilutions, in DMSO, were then made from the 100 mM stock. All DMSO/GC stocks were clear with no visible precipitate.

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Abstract

La présente invention concerne des procédés et des compostions utiles dans le diagnostic et la gestion de maladies auto-immunes. En particulier, la présente invention concerne des procédés et des compositions améliorés pour le diagnostic et la gestion de la maladie de Graves. Les procédés selon la présente invention éliminent non seulement le recours à la radioactivité et sont beaucoup plus simples, économiques, et rapides que les procédés utilisés traditionnellement pour le diagnostic de la maladie de Graves, mais améliorent également les capacités de sensibilité et de détection des dosages pour la détection d'anticorps précédents basée sur la luciférase. De telles améliorations sont basées sur l'efficacité supérieure de dosages comportant un récepteur chimérique TSH en présence d'un glucocorticoïde comprenant, entre autres, le dexaméthasone.
EP08835584A 2007-10-01 2008-09-24 Procédés sensibles et rapides d'utilisation de récepteurs chimériques pour identifier une maladie auto-immune Withdrawn EP2201130A4 (fr)

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AU2008307735A1 (en) 2009-04-09
EP2201130A4 (fr) 2010-10-06
JP5474802B2 (ja) 2014-04-16
CN101918585A (zh) 2010-12-15
KR101560849B1 (ko) 2015-11-20
CA2701198C (fr) 2015-07-21
CA2701198A1 (fr) 2009-04-09
KR20100101072A (ko) 2010-09-16
WO2009045292A1 (fr) 2009-04-09
AU2008307735B2 (en) 2014-04-24
JP2010539975A (ja) 2010-12-24

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