Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • 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
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses

Definitions

• the invention relates to a method for the quantitative determination of human thyroid peroxidase (hTPO) in biological fluids, in culture media, in tissue extracts and in fluids containing highly purified natural hTPO or recombinant hTPO, and a kit for carrying out such a method.
• hTPO human thyroid peroxidase
• Human thyroid peroxidase (designated below as a rule only by the abbreviation hTPO) is a glycosylated haemoprotein which is bound to the thyroid membranes and performs important functions in the biosynthesis of the thyroid hormones.
• the primary structure of the hTPO was determined after various groups had succeeded in cloning the hTPO gene.
• hTPO is important not only because of its actual function in the biosynthesis of the thyroid hormones but also because it has emerged that hTPO is identical to the so-called microsomal antigen which is recognised as the autoantigen of circulating autoantibodies which can be detected in most patients with autoimmune diseases of the thyroid.
• German Patent DE 41 20 412 Cl in which the presence of autoantibodies against hTPO is detected by virtue of the fact that, in the presence of the required antibodies, the formation of a sandwich of a) a first antibody, in particular a monoclonal antibody, b) the antigen hTPO preferably added in crude, natural form and c) a further labelled, preferably likewise monoclonal antibody is disturbed.
• a further labelled preferably likewise monoclonal antibody
• the present invention is based on the surprising finding that it is possible, using two monoclonal antibodies such as those which can be used in the method for the determination of autoantibodies against hTPO according to DE 41 20 412 Cl, to carry out a highly sensitive measurement of the antigen hTPO in various biological fluids and in particular in tissue extracts, in culture media and similar fluids in laboratory practice.
• the present invention therefore relates to a method for the quantitative determination of human thyroid peroxidase (hTPO) in biological fluids, culture media, tissue extracts and fluids containing highly purified natural hTPO or recombinant hTPO, characterised in that the determination is carried out as a sandwich assay known per se, using a first monoclonal antibody against hTPO and at least one further monoclonal antibody against hTPO (MABj .
• hTPO human thyroid peroxidase
• MAB 2 MAB 2 ) , of which the former (MAB recognises hTPO in a region which is sensitive to denaturing and is involved in the binding of the autoantibodies against hTPO and in enzyme inhibition, while the one or more further antibodies (MAB 2 ) recognises hTPO in a region whose binding properties are not impaired by denaturing of hTPO.
• the authors therefore carried out their measurements using a radioimmunoassay in which purified hTPO labelled with 125 I was reacted with a polyclonal rabbit anti-hTPO serum to give immune complexes which were precipitated by the double antibody method by adding a donkey anti- rabbit globulin.
• the degree of displacement of the labelled hTPO from the precipitated immune complexes was determined.
• the method described has a low sensitivity of about 2 ng/ml, and the hTPO serum concentrations determined by the stated method were so high that there is good reason for doubting the correctness of the values obtainable by the stated method.
• the reason for measuring hTPO concentrations in the serum was the desire to test the correctness of various theories on the formation of autoantibodies against hTPO in autoimmune diseases of the thyroid.
• peroxidases play an important role in the metabolism of chemicals and may be substantially involved in the formation of certain metabolites which can be important, for example, for the toxicity of certain substances.
• a reliable method for the direct determination of hTPO can also be used for concentration determination or calibration and standardisation of hTPO contents in commercial products or in products and intermediates obtained by genetic engineering.
• the method according to the invention provides for the first time a method which permits a highly sensitive measurement of the antigen hTPO by the sandwich assay known per se.
• a monoclonal anti-hTPO antibody is fixed, in agreement with the principle known per se, on a solid phase, preferably on the wall of coated polystyrene tubes, and a highly sensitive hTPO assay (lower limit of detection about 26 pg TPO/ l) with highly dynamic characteristics (measuring range more than three orders of magnitude) and high reproducibility (mean interassay coefficient of variance 3.6%) is provided with the use of a further labelled anti-hTPO antibody which is preferably labelled with an acridiniumester and has chemiluminescent properties.
• two antibodies which can also be used in the process according to DE 41 20 412 Cl and which correspond to the antibodies described in J. Ruf et al., Endocrinology 125, and from the clones with the numbers 15 and 53 (referred to below as MAB 15 and MAB 53 for short) are preferably used.
• the two preferably used antibodies were deposited as a precaution, in the form of the hybridoma cells producing these antibodies, in "DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH", Maschenroder Weg lb, Braunschweig, Germany, in accordance with the Budapest Agreement.
• the deposition numbers are DSM ACC2154 (acceptance date 14.09.1993) for the hybridoma with the designation "TPO # 15-4-G2" and DSM ACC2137 (acceptance date 07.07.1993) for the hybridoma "TPO # 53".
• MAB 15 is one of the antibodies which binds hTPO in a region which is also involved in the binding of autoantibodies against hTPO and furthermore in enzyme inhibition.
• the binding of the MAB 15 to hTPO is greatly reduced under conditions which make it likely that denaturing of hTPO will occur, it may be concluded that the stated monoclonal antibody recognises a certain conformation of hTPO (i.e. is a so-called conformation antibody) .
• the MAB 53 J. Ruf et al. , Endocrinology 125
• the MAB 53 preferably used as a further labelled monoclonal antibody is one of the antibodies whose binding is impaired to a very much lesser extent by denaturing of the hTPO, so that it is assumed that this monoclonal antibody tends to recognise a region of the primary amino acid sequence and is therefore a so-called sequential antibody.
• the use of the two stated antibodies leads to an outstanding method for the determination of hTPO, it is however within the scope of the present invention to use one or more labelled monoclonal or polyclonal antibodies with comparable properties instead of the labelled MAB 53 or in addition thereto, i.e. sequential antibodies in the above sense which do not interfere with the binding of hTPO to an MAB 15.
• the MAB 53 can also be used together with one of the monoclonal antibodies 30 to 47 (J. Ruf et al. , Endocrinology 125) or can be replaced by a mixture of the two stated monoclonal antibodies.
• the use of a mixture of two of the stated monoclonal antibodies is of particular interest in connection with the paper by C. de Micco, J.
• Fig. 1 shows the results of the hTPO measurement by the method according to the invention in different measuring matrices
• Fig. 2 shows the specific hTPO values measured by the method according to the invention in the case of different tissue extracts and Fig. 3 shows the effect of the presence of leupeptin on the reproducibility of the hTPO measured in tissue extracts with increasing age of the samples.
• the coupling of the stated monoclonal antibody to a solid phase was carried out by coating a polystyrene tube by a known method, as follows:
• Polystyrene test tubes having dimensions of 12 x 75 mm were each filled with 1 ⁇ g of the anti-hTPO antibody 15 in 300 ⁇ l of an aqueous buffer solution (10 mM tris HC1; 10 mM sodium chloride; pH 7.8). After incubation for 20 hours at room temperature, the tubes were washed twice (4.5 ml of H 2 0 each time) . The tubes were then saturated with a solution of 0.5% BSA (bovine serum albumin) by filling them with the saturation solution, incubating them for two hours at room temperature and then emptying them by decanting the content. The tubes were then freeze-dried with the applied coating. In this form, the ready-to-use tubes were included in the test kit for the method. 2. Preparation of an anti-hTPO antibody labelled with a chemiluminescence label:
• the purified tracer obtained in this manner is diluted to a total activity of 1 x 10 8 RLU (relative light units) per ml in 50 mM Hepes buffer, 1% BSA (from MILES) and 1 mg/ml mouse IgG (from SCANTIBODIES) , pH 6.5, and is filled in portions of 0.5 ml into 10 ml amber glass bottles and then freeze-dried.
• RLU relative light units
• the freeze-dried tubes were included in the kit. Before an anti-hTPO test is carried out, the tracer is reconstituted in each case with 5 ml of a buffer having the following composition: 50 mM Hepes, 100 mM sodium chloride, 0.5% Triton X100 (from PIERCE), pH 6.5, which is also part of the test kit.
• a buffer having the following composition: 50 mM Hepes, 100 mM sodium chloride, 0.5% Triton X100 (from PIERCE), pH 6.5, which is also part of the test kit.
• Various calibrators can be used: a) Very pure thyroid peroxidase, isolated from human thyroid membranes and purified by affinity chromatography (cf. J. Ruf et al. , Endocrinology). b) Recombinant human thyroid peroxidase (purified by affinity chromatography) , which is available from the company WBAG Resources/Zurich, with an hTPO concentration of 36.96 ⁇ g/ml in a buffer solution of 25 mM tris HC1 (pH 7.4) containing 0.1 M KI. c) Crude thyroid peroxidase which is isolated from human thyroid and prepared according to P 41 20 412 Cl and the content of which is calibrated using a) or b) .
• hTPO is diluted to the appropriate concentrations as a five-fold concentrate in PBS (phosphate-buffered saline solution)
• the measurement is carried out by a procedure in which 200 ⁇ l of the standard or of the sample are pipetted into the tubes coated with the immobilised monoclonal antibody and then 100 ⁇ l of tracer are pipetted in each case. After an incubation time of 16 to 20 hours at room temperature under the strict exclusion of light owing to the light sensitivity of the luminescence label, 1 ml of wash solution is added to each tube, after which the tube content is decanted. Washing is then carried out three times with 1 ml of wash solution each time, followed each time by decantation. The tubes are then placed for 5 to 10 minutes with the open side facing downwards on blotting paper in order to suck up the residual fluid. All coated tubes are then placed in a luminometer for measurement of the chemiluminescence signal, the required reagents being added in a known manner, preferably automatically, and the luminous efficiency being measured in a period of 1 s.
• the shape of the standard curve is influenced by the measurement media used, Fig. 1 showing the standard curves obtained for the measurement media buffer, medium + 10% FCS, serum, saliva, plasma and urine.
• the calibrators can be reconstituted in any desired measurement medium, so that there is complete freedom with regard to the choice of the so-called measurement matrix.
• the natural hTPO purified by affinity chromatography and recombinant hTPO purified by affinity chromatography give identical results in the measurements.
• the sensitivity (lower limit of detection of the test) was determined as 26 pg hTPO/ml (mean value of the RLU values of the reference standard plus 3 standard deviations in buffer matrix) , a surprisingly good value .
• hTPO in thyroid tissue extracts, it is also advisable, in order to obtain reproducible results, to carry out the hTPO determination in the presence of an endoprotease inhibitor, in particular of a leupeptin (cf. following Example of Use).
• the samples are frozen on solid carbon dioxide and stored at minus 20°C until required for further use.
• the material is weighed and is homogenised with 10 times the weight of PBS buffer, which additionally contains 0.5% Triton X100 and 500 ⁇ m leupeptin (Ultra-Turrax homogeniser, IKA- Werke/Staufen; 5 times at a maximum speed of 10 s in each case).
• PBS buffer which additionally contains 0.5% Triton X100 and 500 ⁇ m leupeptin (Ultra-Turrax homogeniser, IKA- Werke/Staufen; 5 times at a maximum speed of 10 s in each case).
• the samples are centrifuged for one hour at 100,000 g, and the resulting supernatant solution is measured to determine its protein content and hPTO content. The resulting quotient of the amount of hTPO and the amount of the protein is shown in Fig. 2.
• the specific hTPO content of thyroid tissue or operation materials is subjected to drastic changes. Even with the small number of samples shown, differences in the specific content of up to a factor of 8 are found.
• FIG. 3 shows the results of experiments in which the TPO extract was stored at room temperature with and without the addition of 500 ⁇ M of leupeptin.
• the extracts were measured at different time intervals using TPO standard series. For this purpose, they were diluted in the standard buffer.

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Cited By (5)

Citations (2)

• 1993
  • 1993-12-17 DE DE4343255A patent/DE4343255A1/de not_active Withdrawn
• 1994
  • 1994-05-26 IL IL10979594A patent/IL109795A0/xx unknown
  • 1994-05-31 JP JP7504307A patent/JPH08512407A/ja active Pending
  • 1994-05-31 EP EP94917680A patent/EP0708924A1/de not_active Withdrawn
  • 1994-05-31 AU AU69306/94A patent/AU6930694A/en not_active Abandoned
  • 1994-05-31 WO PCT/EP1994/001771 patent/WO1995002824A1/en not_active Application Discontinuation

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