MXPA97005158A - Monoclonal antibodies anti- - Google Patents

Abstract

The present invention relates to a monoclonal antibody to human thrombopoietin (TPO) with a method for the purification of TPO which comprises isolating the TPO from the TPO-containing material by affinity cormatography, using the antibody, and with a method for quantification or immunochemical detection of TPO using the antibody. Different types of human TPO molecules can be purified, detected or quantified by the use of the anti-T monoclonal antibody

Description

"ANTI-TPO MONOCLONAL ANTIBODIES" BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to an anti-TPO monoclonal antibody useful for the detection, purification or quantification of TPO.

EXHIBITION OF THE RELATED TECHNIQUE Human TPO (thrombopoietin) is a cloned protein as the coordinating group of Mpl which is a member of the cytokine receptor superfamily (from Sauvage et al., Nature (London), volume 369, pages 533 to 565 (1994); T.D. Bartley et al., Cell, volume 77, pages 1117 to 1124 (1994)). Each of these coordinating groups Mpl can be detected in sera and blood plasmas of thrombocytopenic animals (human, mouse, dog) and its involvement in the production of megakaryocytes and platelets has already been confirmed. To develop a therapeutic agent for use in the treatment of thrombocytopenia, the present inventors have purified TPO from the plasma rat of thrombocytopenic rats using, as an indication, an activity that stimulates the production of megakaryocytes from megakaryotic precursor cells. highly purified from rat bone marrow, and have succeeded in cloning the rat TPO cDNA and the human TPO cDNA, based on their partial amino acid sequence and obtaining homogeneous human TPO in a large amount by recombinant DNA technology (H. Miyazaki et al., Exp. Hematol., Volume 22, page 838 (1994)). It has been found that the resulting human TPO has the same amino acid sequence as that of the aforesaid factor obtained as the human Mpl coordinator group (See SEQ ID NO: 1 in the LIST OF SEQUENCES to be noted below). In the context, the superior medium for the detection, purification or quantification of TPO is needed for the production of homogenous human TPO in a large quantity, by means of recombinant DNA technology, for evaluation of the prepared TPO, or for the development of TPO as drugs.

COMPENDIUM OF THE INVENTION The present inventors have studied the preparation of the anti-TPO monoclonal antibodies useful for detection, purification or quantification of TPO, and found that the hybridomas obtained by melting the myeloma cells with antibody-producing cells derived from an animal that has been immunized with the human TPO mutant (referred to as "h6T (1-163)") expressed in Escherichia coli, produced monoclonal antibodies that can recognize h6T (1-163), a human TPO protein that has the amino acid sequence shown in SEQ ID N0: 1 and expressed in CHO cells (referred to as "CHO (1- 332)"), another human TPO protein having an amino acid sequence of positions 1 to 163 shown in SEQ ID NO: 1 and is expressed in CHO cells (referred to as "CHO (1-163)"), etc. and that these monoclonal antibodies could be used for the purification and quantification of human TPO proteins. H6T (1-163) has an amino acid sequence (SEQ ID NO: 2) corresponding to positions 1 to 163 of the amino acid sequence shown in SEQ ID NO: 1, with the exception that the amino acid residues Ser1 and Ala3 are substituted by Ala and Val, respectively, and that the residues of Met and Lys are further fixed to the term N. A process for the production of h6T (1-163) will be illustrated in detail in Example 1 which will be pointed out below. Accordingly, the present invention provides a monoclonal antibody to human TPO. A method for TPO purification which comprises isolating TPO from a TPO-containing material by affinity chromatography, using an anti-human TPO monoclonal antibody, is also provided. The present invention further provides a method for the quantification or immunochemical detection of TPO, which comprises the use of the anti-human TPO monoclonal antibody.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph, showing the change in absorbance of 450/570 nm depending on the dose of CH0. { 1-163) or CHO (1-332). Figure 2 is a graph showing the change in absorbance of 450/570 nm, depending on the content (or concentration) of CHO (1-163), CHO (1-332), MKT (L-163) or MKT ( 1-332). Figure 3 is a graph showing the reactivities of the monoclonal antibody of the present invention with CHO (1-332) and recombinant human erythropoietin, which are compared by the absorbance ratio of A450 / A570.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described below in greater detail. The monoclonal antibody of the present invention can be produced in the following manner. (1) Preparation of immunized animal cells A mouse, rat or animal similar is immunized with a human TPO, from which the spleen cells are prepared. For example, human TPO can be produced in E. coli using recombinant DNA techniques (see Example 1). The immunization is generally carried out by administering from 5 to 100 micrograms of human / animal TPO in combination with an appropriate adjuvant in a mouse, rat or rabbit, subcutaneously, intravenously or intraperitoneally, or in a manner of administration in the plant of the leg. The immunization can be carried out once or several times at appropriate intervals, preferably at intervals of one to two weeks (in the case of administration in the leg plant, from two to three days). Antibody antibody evaluations in the sera of the immunized animals are measured and, based on these results, animals that showed a sufficiently high antibody evaluation are useful as the source of antibody producing cells. Antibody producing cells obtained three to four days after the final immunization are preferred. Various known techniques can be used, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and the like, as methods for measuring an antibody assay, and ELISA is normally used in this invention. (2) Preparation of myeloma cells As myeloma cells, it is generally desirable to use established cells obtained from mice, for example, mouse resistant to 8-azaguanine (BALB / c origin) myeloma cell lines, such as P3 -X63-Ag8-Ul (P3-Ul) [Current Topics in Microbiology and Immunology, 81, 1-7 (1978), P3-NSI / l-Ag4.1 (Ns-1) [European J. Immunoloy, 6, 511-519 (1976). SP2 / 0-Agl4 (SP-2) (Nature, 276, 269-270 (1978)], P3-X63-Ag8.653 (653) (J. Immunology, 123, 1548-1550 (1979)], P3-X63-Ag8 (X63) [Nature, 256, 495-497 (1975)] and the like.These cell lines are subcultured on an appropriate medium, such as an 8-azaguanine medium [8]. -azaguanine plus the normal medium, that is, the RPMI 1640 medium supplemented with glutamine (1.5 mM), 2-mercaptoethanol (5xl0-5 M), gentamicin (10 micrograms per milliliter) and fetal calf serum (FCS, 10 percent) INDM (Dulbecco's Medium Modified with Iscove); or Dulbecco MEM and finally subcultured in or on the normal medium three to four days before cell fusion is carried out so that 2xl07 or more fusion cells can be obtained at the time of fusion. (3) Cell Fusion In the case of mice, for example, 5 to 100 micrograms of the human / animal TPO are administered intraperitoneally or subcutaneously in the mice that have been immunized in the manner of step (1) and then after Three to four days later, the spleens of the mice are removed in order to obtain cells from the spleen. The spleen cells obtained in this way are washed well with a culture medium or PBS as well as the myeloma cells prepared in step (2), after which both cells are mixed in a ratio of spleen cells to myeloma about 5-10:, and then undergo centrifugation. The resulting supernatant fluid is discarded, and the precipitated cells are completely separated and stirred at 37 ° C for one to two minutes, while a solution of polyethylene glycol (PEG: 1,000 to 4,000 molecular weight) in a medium such as DMEM is added to the suspension until that the number of spleen cells reaches 108 cells per 0.1-1.0 milliliter of the suspension, and the situation is continued for one to two additional minutes. After being stirred, 1 to 3 milliliters of the medium is added to the suspension, while stirring for 1 to 3 minutes and subsequently adding 7 to 10 milliliters of the same medium, slowly. The suspension is allowed to stand for 5 to 10 minutes at 37 ° C and then centrifuged. The resulting supernatant fluid is discarded and the remaining cells are resuspended in medium supplemented with FCS and subjected to centrifugation. This procedure is repeated twice or three times. The cells are gently suspended in 50 to 200 milliliters of medium supplemented with FCS and the suspension is distributed in an amount of half the volume of the well in wells of a culture dish and are cultured at a temperature of 35 ° C to 38 ° C. C for 10 to 18 hours using a 3-7 percent CO2 incubator. The HAT medium (hypoxanthine (10 ~ 6 to 10 ~ 3 M), aminopterin (10-8 to 10-7 M) and thymidine (10 ~ 6 to 10 ~ 4 M) in the normal medium) is distributed in an amount of half the volume of the well in the wells of the culture plate, and the cultivation is continued through 10 to 30 hours. Then, at intervals of 10 to 30 hours through one to four days, one-half the volume of the culture supernatant fluid is removed and replaced by the same volume of fresh HAT medium, and the cells are cultured at a temperature of 35 °. C at 38 ° C through 10 to 14 days using a 3-7 percent CO2 incubator. When the hybridoma cells that are grown in the form of a colony are observed in a well, a half volume of the supernatant fluid in the well is removed and replaced by the same volume of an HT medium (HAT medium free of aminopterin), and the replacement of the supernatant fluid by the HT medium is subsequently carried out at intervals of 10 to 30 hours through 1 to 3 days. After 3 to 4 days of culture in the HT medium, a portion of the culture supernatant liquid is taken from the culture and measured for evaluation of the anti-TPO antibody by enzyme immunoassay. The hybridoma, whose productivity of a specific antibody was confirmed by measuring the evaluation of the aforementioned antibody, is transferred to another plate to carry out its cloning. This cloning can be carried out by a method wherein the hybridoma is diluted by limiting the dilution so that a hybridoma cell is contained in a well, a soft agar method where the colony is isolated from a mild agar medium, a method in which a single hybridoma cell is collected using a micromanipulator, and a "clone classifier" method wherein a single hybridoma cell is separated using a cell sorter and the limited dilution method which is simple or easy to handle. As for the steps where the antibody evaluation was confirmed, the cloning was repeated two to four times, for example, by the limitation dilution method, and the clones showing a stable antibody evaluation are selected as the lines of hybridoma cells producing the anti-TPO antibody. (4) Preparation of the monoclonal anti body The hybridomas obtained in the above-mentioned step (3) are cultured in the normal medium instead of the HT medium. A large-scale culture is carried out using a rotating device or rotating bottles. An anti-human TPO monoclonal antibody can be obtained, for example, by subjecting the supernatant fluid obtained from the large-scale culture to a gel filtration to collect and purify the IgG fraction. Alternatively, when the immunized animal is a mouse, the hybridomas can be grown in the abdominal cavities of other mice belonging to similar lines (e.g., Balb / c or Nu / Nu). In this case, when the anti-TPO monoclonal antibody that produces hybridomas that are obtained in step (3) above is administered to female mice treated with pristane (from 4 to 8 born ananas) by intraperitoneal injection in a dose of 4 x 104-107 cells / animal, hybridomas cause the ascites tumor 2 to 3 weeks after administration. When the ascitic fluid is collected from each of the mice and any of the solid components are removed by centrifugation, it can be used as a monoclonal antibody for the detection or quantification and purification of human TPO. If further purification is required, the IgG fraction can be collected from the centrifuged supernatant using the DEAE-Sepharose column, a Protein A-Sephare column or the like. The determination of an isotype or subclass of the antibody is carried out by the method of Ouchterlony (Introduction to Immunological Experiments, Methods for Biochemical Experiments 15, published by Gakki Shuppan Center, Tokyo, Japan, page 74, 1981) or EIA. The protein content is calculated by the Folin-Lowry method by absorbance at 280 nm [1.4 (OD28?) = Immunoglobulin 1 milligram / milliliter]. The methods for the preparation of the monoclonal antibodies of the present invention will be described in the following examples, wherein the monoclonal antibodies (designated L3-1 and L4-1) are obtained from the hybridoma cell lines L3-1 and L4-1 was found to belong to the subclasses IgG 2a and IgG 2b, respectively. The antigen specificities of the monoclonal antibodies obtained L3-1 and L4-1 are described in the following Examples. The monoclonal antibodies of the present invention can be used as coordinating groups in an affinity chromatography for the isolation and purification of human TPO. The immobilized monoclonal antibodies useful in affinity chromatography can be prepared according to various protein immobilization techniques using a gel support such as Sepharose 4B activated with CNBr (Pharmacia Fine Chemicals) or a membrane support. For the purification of human TPO using the immobilized monoclonal antibody, the antibody is packed in a column and a solution containing the human TPO is applied thereto. Then, the unbound substances are washed and the TPO bound to the column is eluted, for example using a glycine-HCl stabilizer (pH 2.5) which may contain sodium chloride, propionic acid, dioxane, ethylene glycol, chaotropic salt, hydrochloride of gluanidine, urea or similar. The monoclonal antibodies of the present invention can be used in the determination of human TPO, for example, by an enzyme immunoassay such as the solid phase sandwich method. In accordance with one embodiment of the present invention, the detection or quantification method of the present invention comprises contacting the anti-TPO monoclonal antibody with a sample containing TPO to detect or quantify an immunological complex formed. The solid phase sandwich method is carried out by the following procedure. The solid phase sandwich method is a type of non-competitive solid phase enzyme immunoassays wherein an antigen is detected or quantified with two different antibodies that can recognize different epitopes of the same antigen. That is to say, an antibody that recognizes TPO is immobilized on a solid phase in the first step, after which in a second step the TPO is absorbed into the solid phase through the formation of a TPO immune complex with the antibody immobilized in the solid phase. In the third step, the TPO is detected or quantified through its reaction with an irradiated antibody or an antibody fragment thereof that can recognize a TPO epitope other than the epitope recognizable by the antibody used in the first step. The irradiated antibody or its fragment can be prepared by irradiating the same with an enzyme such as a strong horseradish peroxidase directly by the maleimide or similar method, or indirectly by the avidin-biotin method or the like.

EXAMPLES The following examples are provided to illustrate the present invention in greater detail.

Example 1 Preparation of an antigenic protein, h6T (I-163) The DNA coding for h6T (1-163) shown in the SEQUENCE LIST (SEQ ID NO: 3) was chemically synthesized and cloned into pCFM536 (ATCC No.39934; JP-A-60-501988) that has been digested with Xbal and HindIII [AND. coli JM109 transformed previously with pMWl (ATCC No.39933) was used as the host]. The clone obtained in this way was named pCFM536 / h6T (1-163) and the strain of E. coli containing this expression vector was used as the transformant to be used in the expression of h6T (1-163). The expression of the expression plasmid pCFM536 is controlled by a? PL promoter which is under the control of a cI857 repressor gene. The transformant described above was cultured overnight at 30 ° C with shaking in 60 milliliters of the LB medium supplemented with 50 micrograms per milliliter of ampicillin and 12.5 microns per milliliter of tetracycline and 25 milliliters of the culture were added to 1,000 milliliters. of LB medium supplemented with 50 micrograms per milliliter of ampicillin and cultured at 30 ° C with shaking from OD to A600 reached 1.0 to 1.2. Subsequently, approximately 330 milliliters of the LB medium preheated to 65 ° C was added so that the final temperature was 42 ° C and the agitation of the culture was continued for three hours at 42 ° C to induce the expression of h6T (1- 163). 0.6 gram of the frozen cells of h6T (1-163) was suspended yielding the transformant in 3 milliliters of water and disintegrated using a high pressure disintegrator. A precipitated fraction was recovered by centrifugation thus removing most of the contaminated proteins, cell components and the like. The precipitated fraction recovered in this way containing h6T (1-163) was suspended in two milliliters of water, mixed with 0.5 milliliter of the stabilizer of 1 M Tris-HCl of pH of 8.5 and 10 milliliters of 10 M of urea and then it was stirred at room temperature for five minutes. To four milliliters of the resulting suspension were added 28 milliliters of the stabilizer of 20 mM Tris-HCl of pH 8.5 and 8 milliliters of the stabilizer of 20 mM Tris-HCl pH of 8.5 containing 1 M of urea, followed by agitation Gentle overnight at 4 ° C. A precipitated fraction was recovered by centrifugation and the precipitated fraction recovered in this manner containing h6T (1-163) was solubilized at room temperature by adding 200 microliters of the stabilizer of 0.5 M Tris-HCl pH of 8.5 containing 8 M guanidine , 5 percent acetonitrile and 0.3 percent EDTA and then underwent one hour of reduction treatment at 60 ° C after the addition of 20 milligrams of DDT. Then, using an elution solvent A (0.1 percent TFA) and an elution solvent B (n-PrOH containing 0.05 percent TFA) a 100 microliter portion of this was applied to Capcell Pak Cl 300A (manufactured by Shiseido Catalogo (Ca.) No. Cl TYPE: SG300A, column of 04.6 x 150 millimeters) equilibrating with 25 percent of B, and then developed a flow regime of 0.4 milliliter per minute with 50 minutes of a linear gradient of 25 percent B to 40 percent B to recover a maximum fraction of h6 (1-163) eluted within the range of about 28 percent to about 30 percent n-PrOH. After the addition of 100 microliters of 50 percent glycerol to this fraction, TFA and n-PrOH were removed by centrifugation-evaporation to prepare h6T (1-163) which was used as the antigen.

Example 2 Preparation of hybridomas that produce anti-human TPO monoclonal antibodies (1) Immunization of mice 500 micrograms of h6T (1-163) obtained in Example 1 are mixed well with 1,000 microliters of the RIDI ADJUVANT (manufactured by IMMUNOCHEM RESEARH, Ca. No. 52017700) to form an emulsion. Then, 5 micrograms / head of the antigen was injected into the paw plant of each Balb / c female mouse (4 weeks old) under etherization. The reinforcers were repeated 8 times in total in the same manner as above at three-day intervals. At the time of the final enhancer, human IL-6 was administered by subcutaneous injection at a dose of 5 micrograms per head. After 3 days of the final booster, the blood was collected from the orbit using a capillary tube of hematocrit and the serum was separated therefrom to measure the titre of the blood antibody, the reactivity with h6T (1-163) by means of a enzyme immunoassay method. Spleen cells from mice that showed 1: 1,000 or more of antibody evaluation and TPO reactivity were used in the subsequent cell fusion. (2) Preparation of myeloma cell line Myeloma cells (P3X63-Ag * 8-653) were thawed, suspended in 10 percent of the DMEM medium added to FCS (manufactured by Hyclone, Ca. No. A-1115-L) and then centrifuged for 300 x grams for 5 minutes. The supernatant fluid was discarded and the remaining cells were resuspended in 10 percent FCS / DMEM and cultured at 37 ° C in a 5 percent CO2 incubator. (3) Cell fusion Cell fusion was carried out in the usual manner. Briefly, the regional lymph nodes (ie, poplieteal, inguinal, and mesenteric) and the spleen of a mouse immunized with h6T (1-163) were aseptically removed and the cells of the lymph node and the spleen cells were suspended separately in the spleen. percent of FCS / DMEM to prepare antibody producing cells. Then, a part of the myeloma cells in the logarithmic growth phase were mixed with 5 parts of the lymph node cells, the cells of the spleen were washed DMEM to remove the serum and then subjected to cell fusion using polyethylene glycol ( PEG 1500, manufactured by Boehringer Mannheim, Ca.NO. 783641). After completing the fusion of the cells, the resulting cells were adjusted to 1x10 ^ cells per milliliter with DMEM containing 10 percent FCS and 10 ng per milliliter of human IL-6 and distributed in 100 microliter portions in wells of a 96-well microtiter plate (Nunc-Immuno Píate MaxiSorp ™, manufactured by InterMed Ca.No. 4-42404). After 18 hours, 10 percent of FCS / 10 ng / milliliter of human IL-6 / DMEM containing 2 x HAT (manufactured by SIGMA, Ca.No.H0262) in a quantity of 100 microliters per well was added to the wells. well, and the cells were cultured through approximately 10 to about 14 days at 37 ° C in a 5 percent CO2 incubator. (4) Selection of hybridomas that produce anti-human TPO monoclonal antibodies Hybridomas producing the anti-human TPO monoclonal antibody were selected by an enzyme immunoassay method. The h6T (1-163) or CHO (1-332) was adjusted to 20 micrograms per milliliter with PBS and distributed in 50 microliter portions in wells of a 96 well microassay plate (Nunc-Immuno Píate MaxiSorp ™, manufactured by InterMed, Ca.No. 4 -42404). The antigen was completely dispersed at the bottom of each well, while the plate was shaken using a microplate mixer (MICROPLATE MIXER MPM-1, manufactured by Iwaki Glass). The plate was sealed with a plate seal (manufactured by SUMILON, Ca.No.MS-30020) and left at 37 ° C for 2 hours or at 4 ° C overnight to observe the antigen towards the plate. Then, after washing with a wash stabilizer of 20 mM Tris-HCl / 0.5 M of NaCl / 0.1 percent Tween 20 (pH 7.5), 300 microliters diluted 4 times of Block Ace was added to each well (manufactured by Dainippon Pharmaceuticals, Ca.No. UK-B25) and the plate was sealed with the plate seal and left at a temperature of 37 ° C for one hour or at 4 ° C overnight. After washing 4 times with the wash stabilizer, 50 microliters of the culture supernatant fluid from a hybridoma clone was added to each well and the plate was shaken using a microplate mixer sealed with the plate seal and then subjected to one hour of reaction at 37 ° C. After completion of the reaction and subsequently 4 times of washing with the wash stabilizer, 50 microliters of alkaline phosphatase irradiated with protein A (PIERCE, Ca.No. 32300) which had been diluted 1: 500 in the diluted Ace Block was added. 10 times and the plate was shaken in a microplate mixer, sealed with the plate seal and then subjected to one hour of reaction at 37 ° C. After the reaction, the wells were washed 4 times with the wash stabilizer, and 100 microliters of a color former from a color development kit for alkaline phosphatase was added (Kirkegaard &; Perry Laboratories, Ca.No. 50-80-00) to each well and the plate was shaken in a microplate mixer, sealed with the plate seal and then subjected to the reaction at room temperature. After about 30 minutes, the absorbance at 405 nm was measured using a plate reading apparatus (ellreader SK601, manufactured by Seikagaku Kogyo). Positive clones for both h6T (1-163) and CHO (1-332) were selected from the wells. The clones selected in this way were expanded and frozen until they were used. At the same time, the cloning was carried out by the limitation dilution method. The limitation dilution was carried out using the 10 percent medium of FCS / 10 ng milliliter of human IL-6 / DMEM, containing lxHT (manufactured by SIGMA, Ca.No.H0137) and a second was carried out. selection in the same manner as above by the enzyme immunoassay method. When the clones were stabilized, they were subcultured in the medium of 10 percent expanded FCS / DMEM and then frozen until used, (5) Recognition site of anti-human TPO monoclonal antibodies By the above method, a total of 63 anti-human TPO monoclonal antibody clones were obtained. Of these clones, 13 clones having high reactivity were selected and their regions that react with the TPO protein were determined by the same enzyme immunoassay method employed in step (4) above, using multiple antigen peptides (HT-1, 2, 3, 4, 5 and 6) (see Table 1) which had been synthesized based on the amino acid sequence of human TPO shown in SEQ ID NO: l. As the results, an antibody that reacts with HT-1 (antibody L3-1), an antibody that reacts with HT-2 (antibody L4-1) and 10 antibodies that react with HT-3 (antibodies Ll-10, S6- 27, S3-13, S2-2, L3-15, L2-43, L3-4, L4-13, S4-12 and S3-52) were obtained.

Table 1 Human TPO (amino acid positions 8 to 28) peptide region HT-1 DLRVLSKLLRDSHVLHSRLSQ Human TPO (amino acid positions 47 to 62) peptide region HT-2 SLGEWKTQMEETKAQD Human TPO (amino acid positions 108 to 126) peptide region HT-3 LGTQLPPQGRTTAHKDPNA Human TPO (amino acid positions 172 to 190) peptide region HT-4 NELPNRTSGLLETNFTASA TPO human (amino acid positions 262 to 284) peptide region HT-5 SLPPNLQPGYSPSPTHPPTGQYT Human TPO (amino acid positions 306 to 332) peptide region HT-6 PSAPTPTPTSPLLNTSYTHSQNLSQEG National Institute of Bioscience and Human-depository Authority: Technology, Agency of Industrial Science and Technology.

Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki- ken, Japan China Authority Center for Type Culture Collection depository: Address: Luo Jia Shan, Wuhan 430072, China.

In this regard, a subclone (L4-1-31) of hybridoma L4-1 and a subclone (L3-1-54) of hybridoma L3-1, were deposited on December 27, 1994 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, under Accession Numbers FERM BP-4956 and FERM BP-4955, respectively. These subclones have also been deposited with the Chinese depositary authority (CCTCC) under Accession Numbers CCTCC-C95003 and CCTCC-C95002, the subclones being called Mouse-Mouse Hybridoma L4-1-31 and Mouse-Mouse Hybridoma L3- 1-54, respectively. (6) Subclass of anti-human TPO monoclonal antibodies The subclass of anti-human TPO monoclonal antibodies L3-1, L4-1, Ll-10, S6-27 and S3-13 obtained in step (5) above were determined by the EIA (INNOGENETICS) method. Each culture supernatant liquid was reacted with an immobilized rat anti-mouse monoclonal antibody (specific subclass) followed by reaction with a rat anti-mouse monoclonal antibody irradiated with alkaline phosphatase (specific to f / light chain) . After completing the reaction, color development was carried out by adding the BCIP substrate to determine a subclass. As the results, subclasses of anti-human TPO monoclonal antibodies L3-1, L4-1, Ll-10, S6-27 and S3-13 were found to be IgG2a, IgG2b, IgG3, IgG3 and IgG3, respectively. (7) Purification of anti-human TPO monoclonal antibodies Hybridomas producing anti-human TPO monoclonal antibodies were cultured in one liter of an eRDF serum-free medium supplemented with 10 milligrams of bovine insulin, 10 milligrams of human transferrin, 20 mM of monoethanolamine and 5 x 10- ^ M of sodium selenite on a large scale using rotating bottles (Falco, Ca.No. 3000). When the survival of the hybridoma reached 90 percent or less, centrifugation was carried out to recover the culture supernatant liquid which was subsequently filtered through a 0.22 micrometer membrane filter. The resulting culture supernatant liquid was applied at a flow rate of 100 milliliters per minute to a Protein A column (manufactured by Fuji Filter Industries K.K., Japan) that had been equilibrated with 0.15 M of a phosphate stabilizer (PBS). After absorption of the antibody from the culture supernatant to Protein A, the unabsorbed substances were washed by passing the PBS through the column at a flow rate of 100 milliliters per minute until the absorption value at 280 nm reached the baseline After washing the non-absorbed substances, the antibody was relieved and recovered with an eluent of 0.1 M glycine (pH 2.5). The recovered solution was neutralized with 0.1 M Tris, adjusted to an appropriate concentration by ultrafiltration, sterilized and then stored at -20 ° C.

Example 3 ELISA using the anti-human TPO monoclonal antibodies The anti-human TPO monoclonal antibody (subclone L3-1-54 of L3-1) which recognizes the HT-1 region as the solid phase antibody was adjusted to 20 micrograms per milliliter with 50 mM of a carbonate stabilizer (pH of 9.2) and distributed in 50 microliter portions to the wells of a 96 well microassay plate (Nunc-I muno Píate MaxiSorp ™, manufactured by InterMed, Ca.No. 4-42404). The solid phase antibody was completely dispersed at the bottom of each well by shaking the plate using a microplate mixer (MIKROPLATE MIXER MPM-1, manufactured by Iwaki Glass). The plate was sealed with a plate seal (manufactured by SUMILON, Ca.No.MS-30020) and allowed to stand at 37 ° C for 2 hours or at 4 ° C overnight to absorb the solid phase antibody towards the plate . After washing with the wash stabilizer (20 mM Tris-HCl / 0.5 M NaCl / 0.1 percent Tween 20 (pH 7.5)), 300 microliters of Block Ace diluted 4 times (Dainippon) was added to each well. Pharmaceuticals, Ca.No. UK-B25) and the plate was sealed with a plate seal and allowed to stand at 37 ° C for one hour or at 4 ° C overnight. After 4 times of washing with the wash stabilizer, 50 microliters of a sample to be tested or a known concentration of TPO as a standard diluted in ACE Block diluted 10 times and the plate was stirred in a mixer were added to each well. The microplate was sealed with the plate seal and allowed to stand at 37 ° C for two hours or at 4 ° C overnight. After the reaction, the wells were washed 4 times with the wash stabilizer, an anti-human TPO monoclonal antibody irradiated with biotin (subclone L4-1-31 of L4-1, primary antibody) which recognizes the HT- region. 2 was diluted to 500 ng per milliliter with Block Ace diluted 10 times and distributed in 50 microlitre portions to the wells, and the plate was shaken in the microplate mixer, sealed with a plate seal and then allowed to stand 37 ° C for 2 hours or 4 ° C during the night. After the reaction, the wells were washed 4 times with the wash stabilizer, avidin irradiated with peroxidase (UltraAvidin ™ - Horseradish Peroxidase, manufactured by Leinco Technologies, Ca.No. A106) was diluted 1: 2,000 in diluted Block Ace. 10 times and was distributed in 50 microlitre portions to the wells, and the plate was shaken in a microplate mixer, sealed with a plate seal and then subjected to a reaction time at 37 ° C. After completion of the reaction and subsequent washing (4 times) with the wash stabilizer, 100 microliters of a color former prepared by adding 1/100 volumes of a substrate solution and a TMBZ color former of one color was added to each well. color development kit for peroxidase (SUMILON, Ca.No.ML-1120T) and the plate was shaken in the microplate mixer, sealed with a plate seal and then subjected to the reaction at room temperature. After about 20 minutes, 100 microliters of a reaction termination solution was added to each well and the plate was shaken in a microplate mixer to stop the staining reaction. Absorbances were measured at wavelengths of 450 nm / 570 nm using an ELISA plate reader (Wellreader SK601, manufactured by Seikagaku Kogyo). A normal curve was made based on the absorption values obtained by known concentrations of CHO (1- 163) or CHO (1-323) (see Figure 1). In addition to CHO (1-163) and CHO (1-332), another normal curve was also made for the human TPO mutants produced in E. coli (eg, "MKT (1-332)" and "MKT ( 1-163) "(see Figure 2) .Therefore, several types of TPO molecules can be measured quantitatively.MKT (1-332) has an amino acid sequence where a Met residue (in position -2) and a residue of Lys (at position -1) are added to the N terminus of the human TPO amino acid sequence shown in SEQ ID NO: 1. MKT (1-163) has an amino acid sequence where a residue of Met (in position -2) and a residue of Lys (in position -1) are added to the N-terminus of amino acid sequence 1-163 shown in SEQ ID NO: 1., the specificity of the antigen was examined using human erythropoietin (hEPO) showing high homology with an N-terminal amino acid sequence of human TPO shown in SEQ ID NO: 1. As a result, it was found that the system can detect TPO specifically (see Figure 3). Also, the detection or quantification of TPO was able to be carried out as in the case of the combination of L3-1-54 and L4-1-31, when the sandwich assay was carried out using a combination of L3-1 and S3-13, S3-13 and L3-1 or S3-13 and L4-1 as the combination of a solid phase antibody and a first antibody irradiated with biotin. As illustrated in the specific examples cited above, different types of human TPO molecules can be detected or quantified by the use of the monoclonal antibodies of the present invention.

LIST OF SEQUENCES INFORMATION FOR SEQ ID NO: l: SEQUENCE CHARACTERISTICS: LENGTH: 332 amino acids TYPE: amino acid ORIGINAL ORIGINAL: Homo sapiens DESCRIPTION OF THE SEQUENCE; SEQ ID NO: l: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val 20 25 30 His Pro Leu Pro Thr Pro Val Leu Leu Pro Val Val Asp Phe Ser Leu 40 45 Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp lie Leu 50 55 60 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln 65 70 75 80 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln 85 90 95 Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu 100 105 110 Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala lie Phe 115 120 125 Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140 Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala 145 150 155 160 Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn 165 170 175 Arg Thr Ser "Gly" TeuT * teu Glu Thr Asn Phe Thr Wing Being Wing Arg Thr 180 185 190 Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys He 195 200 205 Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln He Pro Gly 210 215 220 Tyr Leu Asn Arg He His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe 225 230 235 240 Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp He Ser Ser Gly 245 250 255 Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser 260 265 270 Pro Pro Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu 275 280 285 Pro Pro Thru Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro 290 295 300 Asp Pro Be Wing Pro Thr Pro Thr Pro Thr Pro Pro Leu Leu Asn Thr 305 310 315 320 Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly 325 330 INFORMATION FOR SEQ ID NO: 2: CHARACTERISTICS OF THE SEQUENCE: LENGTH: 165 amino acids TYPE: amineo acid ORIGINAL ORIGINAL: Homo sapiens DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Lys Ala Pro Val Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys -2 -1 1 5 10 Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro 15 20 25 30 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Wing Val Asp Phe 35 40 45 Be Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Wing Gln Asp 50 55 60 He Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg 65 70 75 Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser 80 85 90 Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr 95 100 105 110 Gln Leu Pro Pro Gln Gly Arg Thr Thr Wing His Lys Asp Pro Asn Wing 115 120 125 He Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu 130 135 140 Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr 145 150 155 Thr Ala Val Pro Ser 160 163 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 535 nucleic acids TYPE: nucleic acid TYPE OF CHAIN: double TOPOLOGY: linear TYPE OF MOLECULE: synthetic DNA ORIGINAL ORIGINAL: Homo sapiens DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CTAGAAAAAA CCAAGGAGGT AATAAATA 2 ATG AAA GCA CCT GTA CCA CCT GCA TGT GAT TTA CGG GTC CTG TCT AAA 7 Met Lys Wing Pro Val Pro Pro Wing Cys Asp Leu Arg Val Leu Ser Lys +1 5 10 CTG CTG CGC GAC TCT CAC GTG CTG CAC TCT CGT CTG TCC CAG TGC CCG 12 Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro 15 20 25 30 GAA GTT CAC CCG CTG CCG ACC CCG GTT CTG CTT CCG GCT GTC GAC TTC 17 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Wing Val Asp Phe 35 40 45 TCC CTG GGT GAA TGG AAA ACC CAG ATG GAA GAG ACC AAA GCT CAG GAC 22 Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp 50 55 60 ATC CTG GGT GCA GTA ACT CTG CTT GAG GGC GTG ATG GCT CGC 26 He Leu Gly Ala Val Thr Leu Leu Glu Glu Val Met Wing Wing Arg 65 70 75 GGC CAG CTT GGC CCG ACC TGC CTG TCT TCC CTG CTT GGC CAG CTG TCT 31 Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser 80 85 90 GGC CAG GTT CGT CTG CTG CTC GGC GCT CTG CAG TCT CTG CTT GGC ACC 36 Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr 95 100 105 110 CAG CTG CCG CCA CAG GGC CGT ACC ACT GCT CAC AAG GAT CCG AAC GCT 41 Gln Leu Pro Pro Gln Gly Arg Thr Thr Wing His Lys Asp Pro Asn Wing 115 120 125 ATC TTC CTG TCT TTC CAG CAC CTG CTG CGT GGC AAA GTT CGT TTC CTG 46 He Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu I30 135 140 ATG CTG GTT GGC GGT TCT ACC CTG TGC GTT CGT CGG GCG CCG CCA ACC 50 Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr 145 150 155 ACT GCT GTT CCG TCT TAATGAAAGC TT 53 Thr Ala Val Pro Ser 160

Claims (4)

1. R E I V I N D I C A C I O N E S:
2. A monoclonal antibody to human TPO. 2. A method for the purification of TPO comprising isolating TPO from a material containing TPO by affinity chromatography, using a monoclonal antibody to human TPO.
3. 3. A method for immunochemically quantifying or detecting TPO, characterized by the use of a monoclonal antibody to human TPO.
4. 4. The method according to claim 3, comprising contacting the monoclonal antibody with a sample containing TPO to quantify or detect an immunological complex formed.