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WO1996027129A1 - Immunodosage rapide et automatique de la tsh - Google Patents

Immunodosage rapide et automatique de la tsh Download PDF

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Publication number
WO1996027129A1
WO1996027129A1 PCT/US1996/002593 US9602593W WO9627129A1 WO 1996027129 A1 WO1996027129 A1 WO 1996027129A1 US 9602593 W US9602593 W US 9602593W WO 9627129 A1 WO9627129 A1 WO 9627129A1
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WIPO (PCT)
Prior art keywords
tsh
antibody
sample
region
complex
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PCT/US1996/002593
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English (en)
Inventor
Joel R. L. Ehrenkranz
Original Assignee
Ehrenkranz Joel R L
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Publication date
Application filed by Ehrenkranz Joel R L filed Critical Ehrenkranz Joel R L
Priority to AU49967/96A priority Critical patent/AU4996796A/en
Publication of WO1996027129A1 publication Critical patent/WO1996027129A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Definitions

  • Thyroid stimulating hormone (TSH, thyrotropin) is a 28 kD glycoprotein composed of covalently bound alpha and beta subunits which are produced by the anterior pituitary gland in response to hypothalamic stimulation.
  • TSH regulates the production of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). These hormones have various physiologic effects; primarily, they increase protein synthesis and they increase oxygen consumption in all organs and tissues.
  • Primary hypothyroidism is the decreased secretion of T4, which is part of a negative feedback system that controls TSH production. Reduction in T4 production results in elevated levels of circulating TSH.
  • Primary hypothyroidism is associated with various disease states.
  • TSH Assaying for TSH is a primary screen in the diagnosis of these conditions.
  • Normal TSH levels in neonates i.e., persons up to 4 days post partwri
  • TSH levels in adults i.e., all persons older than neonates
  • the range is 0.3-5 ⁇ lU/mL.
  • fluorometric, chemoluminescent assays, enzyme-linked immunoassays, and radioimmunoassays (RIAs) can measure serum TSH.
  • these require expensive and time- consuming equipment and an infrastructure comprising centralized laboratories and skilled technicians to perform the assays.
  • Congenital hypothyroidism if untreated, can result in irreversible cretinism by six weeks post partum.
  • Centralized laboratories in the industrialized nations annually screen about 12 million neonates for congenital hypothyroidism.
  • Assay results from an on-site laboratory take about 24 hours to obtain; from centralized laboratories, up to about three weeks. Whereas these results are generally available in the industrialized nations, in other parts of the world delays in obtaining assay results and their unreliability can have serious consequences. For example, an estimated 800 million people live in endemic goiter regions, resulting in the birth of about 3.15 million cretins annually. Early, mass screening followed by treatment with synthetic T4, would prevent most of these cases.
  • TRH thyrotropin releasing hormone
  • TRH thyrotropin releasing hormone
  • TRH a cyclic tripeptide hormone secreted by the hypothalamus
  • TRH is commonly administered, i.v. or orally, to patients as a diagnostic tool when there is a question of thyroid disease, since the patient's TSH response to TRH can be measured and this information used to, for example, exclude a diagnosis of hyperthyroidism.
  • the normal response to TRH administration is a prompt rise in serum TSH levels.
  • TSH levels normally rise over a period of 1-180 minutes 5-35 ⁇ lU/mL, with a peak value in 20-30 minutes, maximally at 22+2 minutes.
  • a serum TSH rise in this time period greater than 5 ⁇ lU/mL after the administration of TRH rules out hyperthyroidism.
  • a device which can indicate whether serum TSH levels exceed 5 ⁇ lU/mL can be used to rule out a diagnosis of hyperthyroidism when used in conjunction with TRH administration.
  • the assay comprises a labeled, preferably non- isotopically labeled, antibody in a mobile phase and a second, preferably a different, antibody in a stationary phase.
  • the antibodies are, preferably, chosen such that they are non-competitive; i.e., they bind to different epitopes of, preferably intact, TSH and thus form a "sandwich" complex when both antibodies are bound to the TSH.
  • TSH from the test sample reacts with the mobile antibody and then both are transported to the bound antibody and form a "sandwich" therewith.
  • the TSH assay of the present invention may be used in screening for primary hypothyroidism in neonates and adults. It can also be used in confirming a diagnosis of hypothyroidism or in monitoring the treatment of hypothyroidism. The assay may also be used in monitoring the treatment of thyroid cancer, as hypothyroidism is required for treatment with radioactive iodine. When used in conjunction with TRH administration, the assay may be used to exclude a diagnosis of hyperthyroidism.
  • the kit comprises a substrate divided into four, optionally five, regions.
  • the first region is a reservoir for accepting the aqueous test sample, typically drops of whole blood, although serum or plasma may also be used.
  • Next is a region coated with labeled antibody which has been selected for its high affinity (i.e., Keq ⁇ 1()6) to an epitope of human TSH, preferably intact TSH.
  • this is a colored label such as a colloidal dye or colloidal metal, such as gold, which in concentrated amounts can be seen by the naked eye without the need for instrumentation such as a scintillation counter or spectrophotometer.
  • the antibody and label are not bound to the substrate.
  • the third region contains a second, typically unlabeled, antibody, which is bound to the substrate.
  • This antibody is also selected for its high affinity to human TSH, preferably intact TSH, but, preferably, differs from the first in that it is selective for a different TSH epitope and thus is not competitive with the first antibody.
  • the antibodies used in both regions are preferably monoclonal antibodies.
  • the fourth region acts as a control. This region contains a control reagent that either 1) binds to the labeled antibody, such as TSH or antibody to the labeled antibody; or 2) changes color when hydrated, such as anhydrous copper sulfate.
  • the last region is a sink which absorbs any liquid which has traveled from the reservoir through regions 2-4.
  • the substrate may be one continuous material.
  • the regions may be of different materials; however; each region must be in intimate contact with any adjacent region such that fluid flows, typically by capillary action, unhindered from one region to the next. Each of these regions will be further described below.
  • the antibodies in the second and third regions are chosen for their selectivity to different TSH epitopes.
  • either one or the other may be selected for its affinity to the ⁇ , ⁇ , or ⁇ , ⁇ subunits, with the proviso that at least one has an affinity to ⁇ .
  • both may have an affinity to ⁇ , although this may result in lower assay sensitivity.
  • one antibody is selective for ⁇ subunits and the other for ⁇ , ⁇ subunits.
  • the labeled antibody may be coated on the inside surface of a cup or well which acts as the sample reservoir.
  • the detection zone, the control, and the sink may be on a stick or flat substrate which is dipped into said well after a sample has been placed in the well. In operation the detection zone is below the control but above the sample when the stick is lowered into the sample.
  • the labeled antibody may be added substantially concomitantly with the sample to the reservoir instead of being precoated on the substrate.
  • Preferable is a configuration which comprises a rigid, moisture-impervious solid sheath, generally flat and rectangular in shape, which contains and rigidly holds a substrate in the form of a strip or sheet made of a liquid-conductive material, said material coated with labeled antibody, the bound second antibody, and the control reagent.
  • the sheath is preferably opaque.
  • the upper surface of the sheath contains at least one hole, located over the reservoir end of the substrate, which is used to apply the test sample to the reservoir, and one or two viewing portals, which may either be holes or clear plastic areas, along the center line of said upper surface.
  • the viewing portals are located over the detection zone and the control. If the portals are small, then two portals are used. Alternatively, an elongated single portal which provides visual access to both regions may be used.
  • the upper surface of the sheath may be marked to indicate the detection and control zones.
  • the upper surface of the sheath may also be marked along the detection portal to indicate a range of concentrations when the assay is quantitative.
  • a thin, transparent, moisture-impermeable material may be sandwiched between the substrate and the upper surface of the sheath and the hole over the reservoir may be covered with a removable tight fitting cap.
  • a desiccant such as silica gel may also be contained within the sheath.
  • the sheath may be designed to be less moisture-impermeable if the entire sheath is sealed, together with an optional desiccant, in a moisture-impermeable (e.g., plastic) wrap at the time of manufacture.
  • Devices of this type are commercially available (e.g., Veda Lab, Alencon, France; Solar Care, Bethlehem, PA) and have been described in, e.g., UK Patent GB 2,204,398, which is incorporated herein by reference.
  • Commercially available sheaths are about 7 x 3 x 0.4 cm and contain substrates about 6 x 0.8 cm. Such devices are readily adaptable for use in the present invention.
  • the reservoir can be made from any material capable of absorbing liquid rapidly, preferably within a few seconds of sample application. If commercially available sheaths and substrates as described above are used, the sample size is about four drops (100 ⁇ L) from a pipette.
  • the test sample may also be collected on a fibrous material, such as a cotton swab which is then depressed onto the reservoir.
  • the hole over the reservoir may also be fitted with a scoop- or funnel-shaped cap which can be used to collect blood, as from a finger prick, and convey it to the reservoir.
  • Polypropylene polyethylene, polyvinylidene fluoride, ethylene vinylacetate, acrylonitrile, polytetrafluoro-ethylene, nylon, paper or other cellulosics, such as nitrocellulose, and similar materials may be used. To enhance liquid uptake, these materials may be treated with surfactants to reduce hydrophobicity. Since the sample to be tested may be whole blood, the reservoir also contains a filter which traps red and white blood corpuscles, thus preventing them from migrating into the detection zone.
  • the substrate may be made of the same materials as the reservoir.
  • the substrate is preferably nitrocellulose with a pore size of about 1-12 microns, preferably at least 5 microns, and more preferably 8-12 microns. Nitrocellulose is advantageous in that antibody may be bound to it without prior chemical treatment.
  • binding of the antibody is done using cyanobromide, carbonyldiimidazole, or tresyl chloride.
  • the substrate may be adhered to a backing, preferably made of a thin plastic material. In regions 2-4, the substrate may be sandwiched between two pieces of plastic backing for protection of the reagents thereon.
  • the substrate material is chosen such that liquid migrates through it at a maximum flow rate of about 0.5-0.75 cm/min when the device is in a horizontal position. Further, the spacing between regions 2-4 must be selected such that there is sufficient time for uptake of the labeled antibody in region 2 by the migrating liquid and reaction with the bound antibody in the detection zone. Another timing requirement is that the control should indicate that the assay is complete in 5-10 minutes. Migration rate can be controlled by application to the substrate of surfactants either alone or in combination with viscosity modifiers such as sugars (e.g., sucrose or lactose), cellulosics, or gums (plant or microbial).
  • viscosity modifiers such as sugars (e.g., sucrose or lactose), cellulosics, or gums (plant or microbial).
  • Migration of sample fluid is via capillary action, which can be enhanced by osmotic pressure or gravity.
  • Osmotic pressure is applied by the oncotic pressure of plasma proteins.
  • Gravity can be applied by either constructing the assay kit such that the reservoir is higher than the distal end of the substrate when the kit is placed on a horizontal surface or by instructing the user to position the reservoir higher than the distal end after application of the test sample.
  • the second region of the substrate contains the labeled antibody. Since this antibody must be mobile when exposed to the test sample, it is preferable that it be applied substantially as a surface coating rather than be impregnated into the substrate. This can be achieved by first glazing the second region with compound such as an aqueous solution of sugar, cellulosic, or gum, drying said glaze, and then applying the antibody to the glaze.
  • the antibody applied to the second region is one of a pair of antibodies produced by methods well known in the art which are selected for their high affinity to, preferably intact, TSH and also because, preferably, they bind to different epitopes on, preferably intact, TSH; i.e., their binding is non-competitive.
  • the antibodies should also be selected such that they exhibit little to no cross reactivity with human chorionic gonadotropin (CG), luteinizing hormone (LH), or follicle-stimulating hormone (FSH), and little to no interference from bilirubin, hemoglobin, triglycerides, or plasma proteins. These selections may be made using known protocols to determine cross reactivity or interference with samples containing known amounts of the hormones, proteins, etc. being tested against.
  • the mobile antibody is bound to a label as by covalent or hydrophobic binding such that both antibody and label migrate together into the detection zone.
  • the label may be any readily detectable material. Preferably, it is detectable by the naked eye, such as a colloidal dye, colloidal metal, or colored polymer particles.
  • labels which are detectable by use of optical filters, by stimulation as by UV Of fluorescent light, or by the addition of developing reagents may also be used. These latter, known as indirect labels, are less desirable since they require the addition of the developing reagents, typically enzymes such as alkaline phosphatase, to complete the assay.
  • the preferred label is colloidal gold. Labels of this sort are well known in the art. Colloidal metals are described in EP 7654. Colloidal dyes are described in EP 32270. Colored polymer particles are described in UK 2,204,398.
  • the third region of the substrate, the detection zone contains a bound antibody which complements the mobile antibody, as described above.
  • the substrate in the detection zone it is preferable to impregnate the substrate with antibody in the detection zone to maximize the amount of TSH captured during the assay.
  • the amount of antibody applied in this region determines the intensity of the signal produced during the assay since the labeled antibody in the second region is applied in excess. This amount is determined by calibration against a reference standard containing a known amount of TSH.
  • Both quantitative and semi-quantitative signals can be produced. If a homogenous, narrow band of unlabeled antibody is applied across the width of the substrate, it will produce a uniform color band when exposed to labeled TSH-antibody complex from region 2.
  • a semi-quantitative assay kit can be produced which will detect hypothyroidism in adults or rule out a diagnosis of hyperthyroidism by applying enough antibody in the detection zone to produce a signal only if the test sample contains greater than 5 ⁇ lU/mL of TSH.
  • a semi- quantitative kit can also be produced which will detect hypothyroidism in neonates by applying enough antibody in the detection zone to produce a signal only if the test sample contains greater than 15, preferably greater than 20, ⁇ lU/mL of TSH.
  • the unlabeled antibody is applied with a calibrated concentration gradient, then a series of signals will be produced during the assay.
  • a homogenous band of unlabeled antibody may be applied along the length of an elongated detection zone (e.g., 4 cm of a 6 cm long substrate). Either of these configurations will produce a signal which can be interpreted quantitatively if the portion of the sheath adjacent to the portal overlying the detection zone is calibrated to indicate different levels of TSH, for example, in the ranges 0-25 or 0-40 ⁇ lU/mL.
  • the substrate After impregnation with unlabeled antibody, the substrate is dried and then treated with a blocking agent to block any free binding sites not already bound to antibody.
  • the blocking agent may be one or more of protein, such as bovine serum albumen or casein, polyvinyl alcohol, or ethanolamine.
  • the blocking agent can be applied in excess to the substrate between the second and third regions. During the assay the excess agent will be carried along by the fluid from the test sample and block free binding sites in the detection zone.
  • the fourth, or control, region is designed to indicate that the assay device is working properly.
  • the substrate may be treated with mono- or polyclonal IgG from a non-mouse species (e.g., avian, bovine, equine, etc.), such as unlabeled goat (anti-mouse IgG) antibody, which will react with the labeled murine monoclonal antibody.
  • a non-mouse species e.g., avian, bovine, equine, etc.
  • the control reagent specifically binds with high affinity to the immunoglobulin of the species used to produce the labeled antibodies.
  • the substrate may be treated with TSH, which will react with the labeled antibody.
  • Such reactive materials are preferred, since in addition to merely indicating that fluid from the test sample has traversed regions 2-3, they can minimize false positives which can result from mis ⁇ interpreting weak signals in the detection zone caused by subcritical amounts of TSH being present in the test sample.
  • the user would be instructed to compare the signals in the detection and control zones. The absence of a control signal would indicate a failed assay; i.e., one which is not reliable and should be repeated.
  • the control region may be treated with a substance which will change color when moistened, such as anhydrous copper sulfate. Such a signal generated in the control region is not part of the assay ger se but is a positive control indicating that fluid from the test sample has traversed regions 2-3.
  • the control reagents are preferably bound to the substrate.
  • a sink at the distal end of the substrate is desirable.
  • the sink may be made of any highly absorbent material and may be either an extension of the substrate itself or some other material in contact with the substrate.
  • a nitrocellulose substrate may have attached to it a sink of chromatography paper.
  • the sink should be of sufficient mass that it can continue to absorb whatever liquid reaches it from the reservoir during the five-ten minute period of the assay, thus assuring continued migration of the test sample across the detection zone.
  • the assay described herein is dynamic, i.e., after application of the sample there is constant movement of liquid and components thereof through the various regions of the kit. Some components may remain in one region, travel to an adjacent region, be partially or completely depleted by reaction with another component, etc. This dynamism is inherent in the embodiments of the invention described below. Thus, in step 2 of the assay, "the sample" which migrates from the first region is clearly meant to describe that portion of the applied sample of step 1 which was not retained in the first region.
  • the retained portion may include some liquid and red and white blood corpuscles.
  • the labeled antibody is complexed in region 2 (the labeled antibody having been applied in excess), so that a portion of labeled antibody may also migrate to region 4.
  • One embodiment of the invention is a method of screening for primary hypothyroidism or ruling out a diagnosis of hyperthyroidism which comprises reacting a sample of human blood with labeled antibody with a high affinity to an epitope of, preferably intact, human TSH in a mobile phase to form a TSH -antibody complex and then reacting said complex with an immobilized antibody with a high affinity to an epitope of, preferably intact, human TSH to form a detectable antibody-TSH-antibody complex.
  • Another embodiment of the invention is an immunochromatographic, self-performing assay for measuring, preferably intact, human TSH in the range 0-40 ⁇ lU/mL in an aqueous sample which comprises: 1) applying the sample to a reservoir of a kit which comprises a substrate divided into four, optionally five, regions the first of which is a reservoir for accepting said sample;
  • a second region which is a region coated with unbound, labeled, preferably monoclonal, antibody which has been selected for its high affinity to an epitope of, preferably intact, human TSH and little to no cross reactivity with human chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and little to no interference from bilirubin, hemoglobin, triglycerides, or plasma proteins, whereby labeled antibody-TSH complex is formed;
  • CG human chorionic gonadotropin
  • LH luteinizing hormone
  • FSH follicle-stimulating hormone
  • Another embodiment of the invention is a method of screening for primary hypothyroidism, confirming a diagnosis of hypothyroidism, monitoring treatment of hypothyroidism, monitoring treatment of thyroid cancer, or ruling out a diagnosis of hyperthyroidism which comprises:
  • a sample of human blood to a reservoir of a kit which comprises a substrate divided into four, optionally five, regions the first of which is a reservoir for accepting said sample of human blood; 2) allowing said sample to migrate from said first region through a second region which is a region coated with unbound, labeled, preferably monoclonal, antibody which has been selected for its high affinity to an epitope of, preferably intact, human TSH and little to no cross reactivity with human chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and little to no interference from bilirubin, hemoglobin, triglycerides, or plasma proteins, whereby labeled antibody-TSH complex is formed;
  • CG human chorionic gonadotropin
  • LH luteinizing hormone
  • FSH follicle-stimulating hormone
  • the unbound antibody is non-isotopically labeled. It is also preferred that the sample is human blood. It is also preferred that the antibodies in regions 2 and 3 are different from each other and monoclonal. It is also preferred that one antibody is selective for ⁇ subunits and the other for ⁇ , ⁇ subunits of TSH, preferably intact TSH. It is also preferred that the antibody-TSH-antibody complex is detectable only when the concentration of TSH in the sample is greater than 5 ⁇ lU/mL, or greater than 15 ⁇ lU/mL, or greater than 20 ⁇ lU/mL, or in the range 0- 25 ⁇ lU/mL, or in the range 0-40 ⁇ lU/mL.
  • Another embodiment of the invention is a method as described above wherein the antibody-TSH-antibody complex is detectable only when the concentration of TSH in the sample is greater than 5 ⁇ lU/mL
  • Another embodiment of the invention is a method as described above wherein the antibody-TSH-antibody complex is detectable only when the concentration of TSH in the sample is greater than 15 ⁇ lU/mL, or greater than 20 ⁇ lU/mL
  • Another embodiment of the invention is a method as described above wherein the antibody-TSH-antibody complex is detectable when the concentration of TSH in the sample is in the range 0-40, preferably 0-25 ⁇ lU/mL.
  • the determination comprises: 1) applying the sample of said person's blood drawn after TRH administration to a reservoir of a kit which comprises a substrate divided into four, optionally five, regions the first of which is a reservoir for accepting said sample of human blood;
  • a second region which is a region coated with unbound, labeled, preferably monoclonal, antibody which has been selected for its high affinity to an epitope of, preferably intact, human TSH and little to no cross reactivity with human chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and little to no interference from bilirubin, hemoglobin, triglycerides, or plasma proteins, whereby labeled antibody-TSH complex is formed;
  • CG human chorionic gonadotropin
  • LH luteinizing hormone
  • FSH follicle-stimulating hormone
  • the unbound antibody is non-isotopically labeled. It is also preferred that the antibodies in regions 2 and 3 are different from each other and monoclonal. It is also preferred that one antibody is selective for ⁇ subunits and the other for ⁇ , ⁇ subunits of TSH, preferably intact TSH.
  • kits for screening for primary hypothyroidism which comprises a rigid, moisture- impervious solid sheath containing therein a substrate divided into four, optionally five, regions the first of which is a reservoir for accepting a sample of human blood, the second of which is a region coated with unbound, labeled antibody which has been selected for its high affinity to an epitope of, preferably intact, human TSH, the third of which is a region containing a second antibody, bound to the substrate, which has been selected for its high affinity to an epitope of, preferably intact, human TSH, the fourth of which is optional and contains a control reagent that either 1) binds to the labeled antibody or 2) changes color when hydrated, and the last of which is optional and is a sink which absorbs any liquid which has traveled from the first region.
  • the unbound antibody is non-isotopically labeled and monoclonal. It is also preferred that the antibodies in regions 2 and 3 are different from each other and monoclonal. It is also preferred that one antibody is selective for ⁇ subunits and the other for ⁇ , ⁇ subunits of,. preferably intact, TSH. It is also preferred that the antibody-TSH-antibody complex is detectable only when the concentration of TSH in the sample is greater than 5 ⁇ lU/mL, or greater than 15 ⁇ lU/mL, or greater than 20 ⁇ lU/mL, or in the range 0-25 ⁇ lU/mL, or in the range 0-40 ⁇ lU/mL.
  • kits for measuring, preferably intact, human TSH in the range 0-40 ⁇ lU/mL in an aqueous sample which comprises:
  • a kit which comprises a substrate divided into four, optionally five, regions the first of which is a reservoir for accepting said sample; 2) allowing said sample to migrate from said first region through a second region which is a region coated with unbound, labeled, preferably monoclonal, antibody which has been selected for its high affinity to an epitope of, preferably intact, human TSH and little to no cross reactivity with human chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and little to no interference from bilirubin, hemoglobin, triglycerides, or plasma proteins, whereby labeled antibody-TSH complex is formed;
  • CG human chorionic gonadotropin
  • LH luteinizing hormone
  • FSH follicle-stimulating hormone
  • the unbound antibody is non-isotopically labeled. It is also preferred that the sample is human blood. It is also preferred that the antibodies in regions 2 and 3 are different from each other and monoclonal. It is also preferred that one antibody is selective for ⁇ subunits and the other for ⁇ , ⁇ subunits of, preferably intact, TSH. It is also preferred that the antibody-TSH-antibody complex is detectable only when the concentration of TSH in the sample is greater than 5 ⁇ lU/mL, or greater than 15 ⁇ lU/mL, or greater than 20 ⁇ lU/mL, or in the range 0- 25 ⁇ lU/mL, or in the range 0-40 ⁇ lU/mL.
  • TSH is deposited in a band across the width of nitrocellulose paper (8- 12 ⁇ pore size with plastic backing).
  • Antibody may be obtained from Kara Biologicals, Stanton, N .
  • Nitrocellulose paper may be obtained from Schleicher and Schuell, Keene, N.H. After application of the antibody, the nitrocellulose paper is dried.
  • the nitrocellulose paper is cut into strips approx. 6 x 0.8 cm such that the absorbent pad is at one end of each strip.
  • a strip from step 6 is placed in the bottom half of a 7 x 3 x 0.4 cm plastic sheath.
  • the absorbent pad is overlaid with a blood elements filter (Millipore Coip., Bedford, Mass.).
  • the distal end of the strip beyond the IgG band is overlaid with a piece of bibulous paper (Schleicher and Schuell).
  • a desiccant such as silica gel is also placed in the bottom half of the sheath below the strip.
  • the top half of the sheath is affixed to the bottom half such that a portal suitable for adding test sample overlies the blood elements filter and a portal suitable for viewing overlies the unlabeled antibody band and the IgG band.
  • the top half of the sheath contains means for collecting capillary blood from a puncture wound, as described in U.S.P. 4,397,318, 4,653,512, and 4,646,753.
  • parallel to the viewing portal may be inscribed a scale for determining the amount of TSH which becomes bounded to the unlabeled antibody.
  • the interior of the top half of the sheath may also comprise one or more crimping means which constrain the blood elements filter, the absorbent pad, and the bibulous paper against the elements immediately below them.
  • the amounts of antibodies applied in Steps 1 , 2, and 5 are empirically determined by applying known protocols to one or more samples with known concentrations of TSH and based on the desired range or desired cut-off of assay sensitivity.
  • TSH levels in each pair of samples were measured using both a third generation TSH assay and a rapid TSH assay as in Example 1 calibrated for a 5 ⁇ lU/mL cutoff.

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Abstract

Un dosage de l'hormone de stimulation thyroïdienne (TSH) par immunochromatographie a été mis au point, lequel produit un résultat visible dans les quelques minutes suivant l'application de l'échantillon. Ce dosage comprend un anticorps marqué en une phase mobile, et un deuxième anticorps en une phase fixe. Les anticorps sont choisis de telle sorte qu'ils sont de préférence non compétitifs. En d'autres termes, ils se lient à différents épitopes sur la TSH et forment ainsi un complexe en 'sandwich' dans lequel les deux anticorps sont liés à la TSH. En pratique, la TSH de l'échantillon réagit avec l'anticorps, puis ils sont tous deux transportés vers l'anticorps lié et forment ainsi un 'sandwich' avec ce dernier. Dans un laps de temps assez court, en présence d'une quantité suffisante de TSH dans l'échantillon, la concentration de TSH augmente jusqu'à produire un point visible dans la phase fixe. Le dosage de la TSH, selon l'inventio n, peut permettre de détecter l'hypothyroïdie primaire tant chez le nouveau-né que chez l'adulte. Il peut également être utilisé en association avec l'administration de TRH pour détecter l'hypothyroïdie.
PCT/US1996/002593 1995-02-28 1996-02-26 Immunodosage rapide et automatique de la tsh WO1996027129A1 (fr)

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AU49967/96A AU4996796A (en) 1995-02-28 1996-02-26 Rapid, self-performing tsh immunoassay

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US39580195A 1995-02-28 1995-02-28
US08/395,801 1995-02-28

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WO1996027129A1 true WO1996027129A1 (fr) 1996-09-06

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291190B1 (en) 1998-08-25 2001-09-18 The Board Of Trustees Of The Leland Stanford Junior University Molecular differences between species of the M. tuberculosis complex
US7001775B1 (en) 1998-10-27 2006-02-21 Rsr Limited Assays for autoantibodies
EP2042872A1 (fr) * 2007-09-28 2009-04-01 Sysmex Corporation Test immunologique sandwich et procédé de détection d'un antigène
WO2012166199A1 (fr) * 2011-05-27 2012-12-06 Abbott Point Of Care Inc. Dosages immunologiques de la tsh et procédés de réalisation des dosages immunologiques de la tsh en présence de contaminants endogènes dans des formats de lavage restreints
US9068994B2 (en) 2011-05-27 2015-06-30 Abbott Point Of Care Inc. TSH immunoassays employing scavenging reagents for cross-reacting endocrine glycoprotein hormone analogues
US9199234B2 (en) 2011-05-27 2015-12-01 Abbott Point Of Care Inc. TSH antibodies for point-of-care immunoassay formats

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2204398B (en) * 1987-04-27 1991-08-28 Unilever Plc Analytical test device for use in assays
JPH04108397A (ja) * 1990-08-27 1992-04-09 Internatl Reagents Corp モノクローナル抗体及びそれを用いたｔｓｈ測定法
US5120643A (en) * 1987-07-13 1992-06-09 Abbott Laboratories Process for immunochromatography with colloidal particles
US5141850A (en) * 1990-02-07 1992-08-25 Hygeia Sciences, Inc. Porous strip form assay device method
US5266497A (en) * 1990-08-31 1993-11-30 Japan Synthetic Rubber Co., Ltd. Immunochromatographic assay with improved colored latex

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2204398B (en) * 1987-04-27 1991-08-28 Unilever Plc Analytical test device for use in assays
US5120643A (en) * 1987-07-13 1992-06-09 Abbott Laboratories Process for immunochromatography with colloidal particles
US5141850A (en) * 1990-02-07 1992-08-25 Hygeia Sciences, Inc. Porous strip form assay device method
JPH04108397A (ja) * 1990-08-27 1992-04-09 Internatl Reagents Corp モノクローナル抗体及びそれを用いたｔｓｈ測定法
US5266497A (en) * 1990-08-31 1993-11-30 Japan Synthetic Rubber Co., Ltd. Immunochromatographic assay with improved colored latex

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 118, No. 15, 12 April 1993, (Columbus, Ohio, USA), TAKEOKA et al., "Basic and Clinical Examination on the 'BeriLux TSH' Kit, a Highly Sensitive Chemiluminescent Immunoassay for TSH", page 99, Abstract No. 118:139992y; & TAKEOKA, 1992, 27(4), 985-94 (Japan). *
CLINICA CHIMICA ACTA, 31 October 1991, Vol. 202, No. 3, TUUMINEN et al., "A Rapid Fluorometric Enzyme Immunoassay for the Determination of Neonatal TSH from Blood Spots", pages 167-178. *
DATABASE DERWENT INFO LTD. ON DIALOG, DIALOG INFORMATION SERVICES, INC., (Palo Alto, CA, USA), No. 92-171661/21, KOKUSAI SHIYAKU K.K., "Thyroid-Stimulating Hormone Specific Monoclonal Antibody-for Determination and Purification of TSH by Immunoassay with High Sensitivity and Specificity and no Cross-Reactivity to LH, HCG and FSH"; & JP,A,04 108 397, (July 1992). *
JOURNAL OF IMMUNOLOGICAL METHODS, 1982, Vol. 51, SOOS et al., "Characterization of Monoclonal Antibodies Directed Against Human Thyroid Stimulating Hormone", pages 57-68. *
THYROID TODAY, October/November/December 1990, Vol. XIII, No. 4, SPENCER et al., "Serum TSH Measurement: A 1990 Status Report", pages 1-12. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291190B1 (en) 1998-08-25 2001-09-18 The Board Of Trustees Of The Leland Stanford Junior University Molecular differences between species of the M. tuberculosis complex
US6686166B2 (en) 1998-08-25 2004-02-03 The Board Of Trustees Of The Leland Stanford Junior University Molecular differences between species of the M. tuberculosis complex
US7364740B2 (en) 1998-08-25 2008-04-29 The Board Of Trustees Of The Leland Stanford Junior University Molecular differences between species of the M. tuberculosis complex
US7700118B2 (en) 1998-08-25 2010-04-20 The Board Of Trustees Of The Leland Stanford Junior University Molecular differences between species of the M. tuberculosis complex
EP1108060B1 (fr) * 1998-08-25 2010-04-28 The Board Of Trustees Of The Leland Stanford Junior University Differences moleculaires entre des especes du complexe m. tuberculosis
US7001775B1 (en) 1998-10-27 2006-02-21 Rsr Limited Assays for autoantibodies
EP2042872A1 (fr) * 2007-09-28 2009-04-01 Sysmex Corporation Test immunologique sandwich et procédé de détection d'un antigène
CN103649755A (zh) * 2011-05-27 2014-03-19 雅培医护站股份有限公司 Tsh免疫测定以及用于在存在内源性污染物的情况下以有限洗涤形式进行tsh免疫测定的方法
WO2012166199A1 (fr) * 2011-05-27 2012-12-06 Abbott Point Of Care Inc. Dosages immunologiques de la tsh et procédés de réalisation des dosages immunologiques de la tsh en présence de contaminants endogènes dans des formats de lavage restreints
US9068994B2 (en) 2011-05-27 2015-06-30 Abbott Point Of Care Inc. TSH immunoassays employing scavenging reagents for cross-reacting endocrine glycoprotein hormone analogues
US9199234B2 (en) 2011-05-27 2015-12-01 Abbott Point Of Care Inc. TSH antibodies for point-of-care immunoassay formats
US9201078B2 (en) 2011-05-27 2015-12-01 Abbott Point Of Care Inc. TSH immunoassays and processes for performing TSH immunoassays in the presence of endogenous contaminants in restricted wash formats
CN103649755B (zh) * 2011-05-27 2016-03-02 雅培医护站股份有限公司 Tsh免疫测定以及用于在存在内源性污染物的情况下以有限洗涤形式进行tsh免疫测定的方法
US9575077B2 (en) 2011-05-27 2017-02-21 Abbott Point Of Care Inc. TSH antibodies for point-of-care immunoassay formats
US9575076B2 (en) 2011-05-27 2017-02-21 Abbott Point Of Care Inc. TSH immunoassays and processes for performing TSH immunoassays in the presence of endogenous contaminants in restricted wash formats
EP2715368B1 (fr) 2011-05-27 2018-02-14 Abbott Point Of Care, Inc. Dosages immunologiques de la tsh et procédés de réalisation des dosages immunologiques de la tsh en présence de contaminants endogènes dans des formats de lavage restreints
EP2715368B2 (fr) † 2011-05-27 2021-01-20 Abbott Point Of Care, Inc. Dosages immunologiques de la tsh et procédés de réalisation des dosages immunologiques de la tsh en présence de contaminants endogènes dans des formats de lavage restreints

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