METHOD FOR THE DETERMINATION OF HORMONES

Abstract: Described is an immunoassay method which makes it possible to determine the amounts of thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone and human choriogonadotropin with the substantially same degrees of sensitivities for all the hormones, in which there is used a monoclonal antibody derived from a hybridoma obtained using the spleen cells of an animal immunized with only one of the hormones or its α-subunit.

Description: METHOD FOR THE DETERMINATION OF HORMONES Background of the Invention: The present invention relates to a method for the determination of thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorigonadotropin (HCG), based on an nanunoassay technique by using monoclonal antibodies. Determination of the amounts of these hormones contained in body fluids, particularly in blood, is extensively utilized in diagnosis of such disease as Graves disease, cetrinism or hypothyroidism, in which the determination is conducted through immunoassay such as EIA (enzyme immunoassay) or RIA (radioimmunoassay) by using antibodies specific for the constituents of the hormones. Each of the hormones is constituted of its own -subunit and B-subunit, in which the antigenicities of the a-subunits of the four hormones are quite similar to each other. Thus, conventional immunoassay techniques (EIA, RIA or the like), in which polyclonal antibodies are used, suffer from poor specificities and reproducibilities, due to the cross reactions between the hormones. For the purpose of overcoming such disadvantage, new immunoassays have been proposed in which there are used monoclonal antibodies specific for the respective hormones. For example, "Clinical Chemistry, Vol. 28, No.9, pp. 1862 1866, 1982" by H. G. Wada et al. discloses EIA which employs monoclonal antibodies specific for -subunit and ss-subunits of the above-mentioned hormones. More specifically, in the disclosed EIA, the anti-a-subunit monoclonal antibodies are used as the solid-phase antibodies (the capture antibodies) for the hormones and the anti-ss-subunits monoclonal antibodies are coupled to a peroxidase to be used as the enzyme-labelled antibodies (the enzyme-antibody conjugate). Mention is made in H.G. Wada et al. that the anti-a-subunit monoclonal antibodies as the solid-phase can only be a monoclonal antibody specific for one of the a-subunits of the hormones because of the similarity in structures and immunochemical properties between the a-subunits of these hormones. However, the experiments conducted by the present inventors in the manner disclosed by Wada et al. proved that such an anti--subunit monoclonal antibody does not necessarily exhibit the same degree of reactivity for all the hormones and considerablyvariesdepending upon the clones from which the monoclonal antibodies are prepared. Thus, it is desired to prepare a monoclonal antibody which makes it possible to determine the amounts of the four hormones with substantially the same degree of sensitivity. Summary of the Invention: A primary object of the present invention is therefore to provide an improved immunoassay for the determination of TSH, FSH, LH and HCG with high sensitivity by use of a monoclonal antibody. For accomplishing the above object, the present inventors have made full studies of monoclonal antibodies, which can be derived fram the fusion of animal spleen cells, preferably mouse spleen cells irranunized with one of the hormones or its i-subunit and myeloma cells, and found that such monoclonal antibodies are classified in to several groups in terms of their reactivities with the hormones, depending upon the clones from which the antibodies are produced. For example, it has been found that monoclonal antibodies from the fusion of mouse spleen cells immunized with TSH (TSH-whole) or its asubunit and myeloma cells can be classified into the following four groups. (a) A monoclonal antibody having distinctly lower reactivity with LH than with the other hormones. (b) A monoclonal antibody having distinctly higher reactivity with LH than with the other hormones. (c) A monoclonal antibody having high reactivites with TSH and LH but low reactivities with HCG anf FSH. (d) A monoclonal antibody having high reactivites with all the four hormones. This fact probably accounts for the presence of at least four epitopes in the a-subunit of the hormone. Thus, for determining the amounts of all the four hormones with high sensitivities it is essential to establish and use such a monoclonal as classified in (d). The present invention is based on the above finding and provides a method for the determination of TSH, FSH, LH and HCG by means of immunoassay, which comprises using, as the solid-phase antibody (the capture antibody) or as the labelled antibody (the enzyme-labelled antibody, the radioactively labelled antibody or the like) for the assay, a monoclonal antibody having been produced from the cells immunized with one of the four hormones or its a-subunit and having high reactivites with all the hormones. The monoclonal antibody to be used in the present invention can be prepared form the fusion of mouse spleen cells immunized with one of the four hormones or its asubunit and myeloma cells.. The preparation of monoclonal antibodiesis carried out in a conventional manner known to those skilled in the art, as will be described below. Among monoclonal antibodies produced from various clones are selected monoclonal antibodies having high reactivities with all the hormones, which reactivities can be expressed in terms of the B/T value (as defined later) and are preferably at least 40 % (with respect to the original solution which is also defined later)k for the effective immunoassay. The features of the present invention will be apparent from the following description, which is only illustrative and not restrictive for the present invention. Preparation of monoclonal antibodies: A W/O suspension for use as antigen for primary immunization. is prepared by dissolving 50 ug of TSH (TSH whole)or the a-subunit of TSH in 0.9 % physiological saline, and adding to the resultant solution 1.3 ml FCA (as adjuvant) available from DIFCO Corp.. A booster antigen is prepared by dissolving 50 ug of TSH (TSH-whole) or TSH asubunit in 0.9 % physiological saline. Female BALBIc mice, at the ages of four to five weeks, are immunized in accordance with the schedule as shown in Table 1. The immunized mouse spleen cells are fused with mice myeloma cells (e.g. P3-X63-Ag8-U1) by means of the cell fusion technology for example, as shown in "Hybridoma Process and monoclonal antibodies" edited by T. Watanabe, pp. 20 - 29, R & D planning Corp. (Japan), 1982. followed by cloning to obtain six monoclonal antibodies. Table 1 Immunization Schedule Clone Antigen Primary Immunization Booster Immunization Cell fusion NO. Amount Injecton Time of injection Amount Injection 1 TSH-&alpha; 5 g + FCA i.p. 48 days 5 g i.v. Four days after (after primary Booster immunization immunization) 2 TSH do. do. 60 days 10 g do. do. 3 TSH do. do. 48 days 5 g do. do. 4 TSH do. do. do. do. do. do. 5 TSH-&alpha; do. do. do. do. do. do. 6 TSH do. do. 60 days 10 g do. do. Clonal specificities of the six monoclonal antibodies were determined with the RIA method by using 125I-labelled TSH, 125I-labelled a-subunit TSH and 125I-labelled 13-subunit TSH as the tracers, with the results given in Table 2, in which the values for the specificities are expressed in cpm as compared with the negative controls of the culture supernatants from the mouse myeloma cells (P3-X63-Ag8-U1). Table 2 Clone Tracer Antigenic No. TSH-whole a ss Specificity 1 4.9 21.2 1.0 a 2 6.2 32.1 1.1 a 3 3.3 6.6 1.0 a 4 6.5 33.8 1.0 a 5 5.7 17.7 1.4 a 6 9.3 42.1 1.2 a The monoclonal antibodies were further determined with respect to the reactivities with the four hormones, the association constants to TSH, and the immunoglobulin subclass. In determining the reactivities, an original solution for each monoclonal antibody was prepared by adjusting the immunoglobulin concentration in the culture supernatant of the clone to be 20 Vg/ml. 100 ug of such original solution for each monoclonal antibody and 100 ul of its dilute solution (diluted five times) are reacted with 125I-labelled TSH, 125I-labelled HCG, 125I-LH or 125I-FSH as the tracer (each 100 ul) to determine the reactivities (B) expressed in cpm (counts per minute), as shown in Table 3. In the table are also provided the ratios B/T, in which T designates the total counts (cpm) of each tracer (100 pal). The association constants of the monoclonal antibodies were obtained by examining the ascites fluids containing the clones, as followes in the known manner, for example, as described in "Experimental procedures in immunoloagy IX - Determination of association constant of antibodies" published by immunological Society of Japan on December 4, 1980, pages 269 - 2705: The monoclonal antibodies were also determined with respect to the immunoglobulin-subclass, through Ouchterlony's method, by examining the culture supernatants from the respective clones, by use of antiserum containing antibodies against mouse-a, -y1, -y2a, -y2b, -y3, Z or -X chain. The results are summarized in Table 3. It is particularly noted that there were obtained monoclonal antibodies having different reactivites which can be classified into the four groups as described precedently. Of the monoclonal antibodies obtained, the antibody of clone No.6 has high reactivities with all the four hormones, with all the B/T values being at least 40 %, and belongs to the group (d). Noticably the clone No.6 appeared to be of an immunoglobulin subclass which is quite similar to IgM, while all the other clones are of IgG immunoglobulin subclass. Table 3 EMI10.1 <SEP> Reactivity <SEP> with <SEP> each <SEP> hormone <SEP> Association <SEP> Immunoglobulin <tb> Clone <SEP> TSH <SEP> HCG <SEP> L@ <SEP> FS@ <SEP> Reac- <SEP> constant <SEP> subclass <tb> No. <SEP> Original <SEP> 1/5 <SEP> Original <SEP> 1/5 <SEP> Original <SEP> 1/5 <SEP> Original <SEP> 1/5 <SEP> tivity <tb> <SEP> solution <SEP> diluted <SEP> solution <SEP> diluted <SEP> solution <SEP> diluted <SEP> solution <SEP> diluted <SEP> group <SEP> (liters/mole) <SEP> H <SEP> chain <SEP> L <SEP> chain <tb> <SEP> B(cpm) <SEP> 5960 <SEP> 4820 <SEP> 5592 <SEP> 5249 <SEP> 1657 <SEP> 1204 <SEP> 4267 <SEP> 3080 <tb> 1 <SEP> a <SEP> 4.5 <SEP> x <SEP> 108 <SEP> &gamma;1 <SEP> K <tb> <SEP> B/T(%) <SEP> 29.2 <SEP> 23.6 <SEP> 28.8 <SEP> 27.1 <SEP> 9.2 <SEP> 2.7 <SEP> 23.0 <SEP> 16.6 <tb> <SEP> B(cpm) <SEP> 5347 <SEP> 3882 <SEP> 5764 <SEP> 4460 <SEP> 1310 <SEP> 906 <SEP> 4164 <SEP> 2845 <tb> 2 <SEP> a <SEP> 4.0 <SEP> x <SEP> 108 <SEP> &gamma; ;1 <SEP> K <tb> <SEP> B/T(%) <SEP> 26.2 <SEP> 19.0 <SEP> 29.7 <SEP> 23.0 <SEP> 7.2 <SEP> 5.0 <SEP> 22.4 <SEP> 15.3 <tb> <SEP> B(cpm) <SEP> 3532 <SEP> 1769 <SEP> 5222 <SEP> 3882 <SEP> 10539 <SEP> 8485 <SEP> 1784 <SEP> 731 <tb> 3 <SEP> b <SEP> 3.0 <SEP> x <SEP> 108 <SEP> &gamma;1 <SEP> K <tb> <SEP> B/T(%) <SEP> 17.3 <SEP> 8.6 <SEP> 26.9 <SEP> 20.0 <SEP> 58.3 <SEP> 46.9 <SEP> 9.6 <SEP> 3.9 <tb> <SEP> B(cpm) <SEP> 9933 <SEP> 8387 <SEP> 6131 <SEP> 4948 <SEP> 10754 <SEP> 9179 <SEP> 4824 <SEP> 3528 <tb> 4 <SEP> c <SEP> 3.8 <SEP> x <SEP> 109 <SEP> &gamma;1 <SEP> K <tb> <SEP> B/T(%) <SEP> 48.6 <SEP> 41.0 <SEP> 31.6 <SEP> 25.5 <SEP> 59.5 <SEP> 50.8 <SEP> 26.0 <SEP> 19.0 <tb> <SEP> B(cpm) <SEP> 9093 <SEP> 7273 <SEP> 4890 <SEP> 4152 <SEP> 8569 <SEP> 6348 <SEP> 3654 <SEP> 2925 <tb> 5 <SEP> c <SEP> 6.1 <SEP> x <SEP> 109 <SEP> &gamma; ;1 <SEP> K <tb> <SEP> B/T(%) <SEP> 44.5 <SEP> 35.6 <SEP> 25.2 <SEP> 21.4 <SEP> 47.4 <SEP> 35.1 <SEP> 19.7 <SEP> 15.8 <tb> <SEP> B(cpm) <SEP> 8605 <SEP> 6770 <SEP> 7819 <SEP> 6481 <SEP> 9307 <SEP> @123 <SEP> @500 <SEP> 5825 <SEP> similar <tb> 6 <SEP> d <SEP> 1.2 <SEP> x <SEP> 109 <SEP> to <SEP> @@ <SEP> K <tb> <SEP> B/T(%) <SEP> 42.1 <SEP> 33.1 <SEP> 40.3 <SEP> 33.4 <SEP> 51.5 <SEP> 44.9 <SEP> 45.@ <SEP> 31.4 <tb> Negative <SEP> 875 <SEP> 910 <SEP> 654 <SEP> 408 <tb> Control <SEP> 20438 <SEP> 19403 <SEP> 18076 <SEP> 18556 <tb> Total <SEP> (cpm) <tb> Example EIA experiments were carried out by using the six clones (monoclonal antibodies) prepared in the manner as described above. A standard solution (200 ul) having a predetermined concentration of one of the hormones and polystylene beads coated with one of said antibodies (the solid-phase) are placed in a tube so as to make the reaction therebetween at 37 OC for 2 hours. The beads were then washed three times with 5 % Tween 20 - PBS. There was added 200 ;l of an anti-B- subunit antibody for the reaction at 37 C for 2 hours, followed by washing three times with 5 % Tween 20 - PBS. To the washed product was added 500 ul of a solution which has been obtained by dissolving 25 Vl of 5 % H2O2 and 15 mg of o-phenylenediamine 2HCl in 25 ml solution containing 0.1 M citric acid and 0.2 M sodium phosphate, so as to carry out the reaction at 37 OC for one hour in dark. The reaction was terminated by the addition 125 ul of 6N H2SO4.A standard calibration curve for each assay system was obtained by plotting the optical densities (O.D.) of the reaction products (in terms of absorbances at 492 nm) agaubst the concentraiton of each hormone. As the sensitivity of each assay system was regarded the concentration value corresponding to the O.D. at the zero concentration multiplied by 2.1. Thus, the value signifies the minimum concentration which can be detected by the assay system. The results are summarized in Table 4. The hormones used in the preparation of the standard solutions were those available from Calibiochem (U.S.A.) and had the following activities: Hormone Activity TSH 5 I.U./ml HCG 6,610 I.U./ml LH 10,500 I.U./ml FSH 5,800 I.U./ml Table 4 Assav sensitivities (in ng/ml) EMI12.1 <tb> Clone <SEP> No. <SEP> TSH <SEP> HCG <SEP> tH <SEP> FSH <tb> <SEP> 1 <SEP> 0.1 <SEP> - <SEP> 0.2 <SEP> 0.2 <SEP> @ <SEP> 1.6 <SEP> 0.2 <SEP> - <SEP> 0.4 <tb> <SEP> 2 <SEP> 0.1 <SEP> - <SEP> 0.2 <SEP> 0.2 <SEP> 1.6 <SEP> - <SEP> 3.2 <SEP> 0.2 <SEP> - <SEP> 0.4 <tb> <SEP> 3 <SEP> 0.4 <SEP> 0.2 <SEP> - <SEP> 0.4 <SEP> 0.025 <SEP> 1.6 <tb> <SEP> 4 <SEP> 0.025 <SEP> 0.2 <SEP> 0.025 <SEP> 0.2 <tb> <SEP> 5 <SEP> 0.025 <SEP> 0.2 <SEP> - <SEP> 0,4 <SEP> 0.05 <SEP> 0.4 <tb> <SEP> 6 <SEP> 0.025 <SEP> 0.05 <SEP> 0.05 <SEP> 0.05 <tb> As can be seen from the results given in Table 4, the use of the clone No.d6 provides an immunoassay which makes it possible to determine all the four hormones with very high sensitivities. Thus, according to the present invention there is provided a method for determining the four hormones by use of a single monoclonal antibody,and the solid-phase antibidy or the labelled antibody for immunoassay, which can be produced by the immunization of mice with only one of the hormones and its a-subunit and have high sensitivities with all the hormones. A corresponding labelled-antibody to be used with the solid-phase of said highly reactive monoclonal antibody in an iirnunoassay is preferably an anti-ss-subunit monoclonal antibody produced by the immunization with the same hormone (as used to prepare the solid-phase antibody) or its B-subunit. Similar remarks would apply to a corresponding solid-phase antibody when the above-mentioned highly reactive monoclonal antibody is used as the labelled antibody. Examples of such anti-B-subunit monoclonal antibodies are described in the applicant's co-pending application entitled "Method for determination of TSH". Further, anti-ss-subunit monoclonal antibodies as described in H. G. Wada et al. can also be used. Although the present invention has been described with reference to preferred embodiments, it has to be understood that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. For example, the method of the present invention is not restricted to EIA as described in the Example, but can be applied also to other immunoassays (e.g. RIA) as well.

Claims: Claims 1. A method for the determination of thyroidstimulating hormone, follicle-stimulating hormone, luteinizing hormone and human chorigonadotropin by means of immunoassay, which comprises using, as the solid-phase antibody or as the labelled antibody, a monoclonal antibody produced by a hybridoma derived from spleen cells of an animal immunized with one of said hormones or its -subunit and having high reactivities with all of said hormones. 2. The method as claimed in claim 1 in which the monoclonal antibody has a reactivity of at least 40 % in terms of the B/T (with respect to the original solution) as defined in the specification, with all of said hormones. 3. The method as claimed in claim 1 or claim 2 in which the monoclonal antibody is produced by a hybridoma derived from spleen cells of a mouse immunized with one of said hormones or its -subunit fused with myeloma cells. 4. The method as claimed in claim 3 in which the monoclonal antibody is produced by a hybridoma derived from spleen cells of a mouse immunized with TSH or the > -subunit of TSH fused with myeloma cells.

Inventors: EDA,YASUYUKI, FUJITA, HARUO, NAKASHIMA, TOSHIHIRO, TSUJI, ICHIROH, UNOKI, MASANORI
Applicants: JURIDICAL FOUNDATION THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE
Application year: 1985
Main classification: G01N33
IPC codes: A61K39, G01N33, C12P21, C07K16, C12N15