JPH04232461A - Measuring method using antiactibin antibody - Google Patents

Abstract

PURPOSE:To predict and diagnose abnormality of a megakaryocyte-platelet system by using an antiactibin and an antibody to an actibin-like substance as a solid phasing antibody and marker antibody, and qualifying and quantifying according to the solid phase sandwich technique or the like. CONSTITUTION:This measuring method uses an antibody to recognize actibin and part of actibin-like substances. The antibody is required to recognize one or two or more kinds of actibin, etc., and preferably part of actibin A. In the first place, an animal is turned to be immune with using actibin or the like as an immunogen. The animal effective as an immunogen is an animal with hot blood such as a rabbit, a chicken, etc. When the antibody is polyclonal antibody, antiserum obtained from the immunized animal is precipitated by saturated ammonium sulfate and immunoglobulin G fraction is separated. It is then judged whether an antiactibin or the like is present in the fraction. A substance to be detected in a sample is trapped through the antigen-antibody reaction with the obtained antibody. The detection of the substance is carried out, e.g. by the agglutination, sandwich technique, etc.

Description

Description: [0001] The present invention relates to a method for measuring activin and/or an activin-like substance, in particular a method comprising using an antibody that recognizes activin and/or an activin-like substance. Regarding measurement methods. activin and/or
Alternatively, the results obtained by measuring activin-like substances provide useful information for predicting and diagnosing increases, decreases, and/or functional abnormalities in the megakaryocyte-platelet system, as well as for predicting and diagnosing diseases accompanied by these. do. [0002] In the blood circulating in a living body, there are cells such as red blood cells, white blood cells, lymphocytes, and platelets, each of which has a unique function to maintain the homeostasis of the living body. is in charge of Among these blood cells, erythropoietin, G-CSF, GM-CSF, M-CSF,
It is known that factors such as various interleukins are involved, and some of them are beginning to be applied as pharmaceuticals. However, research on factors involved in the differentiation and maturation of the megakaryocyte-platelet system (tentatively named thrombopoietin) has lagged behind compared to other blood cell systems. [0003] Only recently has it been reported that IL-6 promotes the maturation of megakaryocytes, which are precursor cells of platelets (Toshiyuki Ishibashi et al.
tal. , Proc. Natl. Acad.
Sci. USA, Vol. 86, p. p. 5953
-5957, 1989; Toshiyuki I
Shibashi et al. , Blood, V
ol. 74, p. p. 1241-1244, 198
9). However, there is no clear causal relationship between the blood concentration of IL-6 and the platelet count, and
It is known that L-6 exhibits a wide variety of actions and is deeply involved in the induction of inflammation as an acute phase reaction protein in vivo, so it is believed that IL-6 is a physiological thrombopoietin. has not been considered. [0004] Therefore, substances other than the above-mentioned IL-6 promote the differentiation, maturation and/or proliferation of megakaryocyte-platelet cells, and there is a difference between the blood concentration and the number of platelets and/or megakaryocytes. It is desired to identify true thrombopoietin-like physiologically active substances that have a causal relationship and to apply them to pharmaceuticals and diagnostic reagents. On the other hand, activin is a substance purified from pig follicular fluid in 1986 as a substance that promotes the secretion of follicle-stimulating hormone. And SDS-PAGE
As a result of analysis of the amino acid sequence of the N-terminus, activin is
A homodimer of the βA chain (Wylie Vale et al., Na
ture, Vol. 321, p. p. 776-77
7, 1986) or a heterodimer of βA and βB chains (Nicholas Ling et al.
, Nature, Vol. 321, p. p. 77
9-782, 1986). And already, the βA chain and βB chain of human activin
The amino acid sequence and gene sequence of the chain have also been revealed. As mentioned above, it has been confirmed that there are at least two types of activin molecules, and activin, which consists of a homodimer of βA chains, is activin A and βA.
Activin consisting of a heterodimer of βB chains is called activin AB. Activin consisting of a homodimer of βB chains is called activin B, and is thought to exist in nature, but its existence in nature has not yet been confirmed or published. however,
All three types of activins mentioned above have been produced using genetic engineering techniques, and all have been recognized to have follicle-stimulating hormone secretion promoting activity (Hiroko Fujita et al., Experimental Medicine, Vol. 8).
pp. 314-320, 1990, and Mason
A. J. et al. , Molecular E
ndocrinology, vol. 3, p. p
．． 1352-1358, 1989). By the way, in addition to promoting the secretion of follicle-stimulating hormone, these activins can also be used to treat autoimmune diseases,
It is expected to be used as a promoter of tissue regeneration (European Patent Publication No. 368992). In addition, activin A is derived from EDF (Erythroid differencium).
(Masahiro Murata)
et al. , Proc. Natl. Acad
．． Sci. USA. , Vol. 85, p. p.
2434-2438, 1988), EDF was originally found in the culture supernatant of a human leukemia-derived cell line as a substance that has the activity of differentiating Mouse Friend leukemia cells into red blood cells. Activin A (EDF) has an effect on the human erythroid leukemia cell line K-5 in addition to the above-mentioned action on mouse friend leukemia cells.
It has also been confirmed that 62 has the activity of differentiating into red blood cells (John Yu et al., Natu
re, Vol. 330, p. p. 765-767, 1
987). [0008] Thus, activin A is
Activin A is expected to differentiate and mature leukemic cells into normal cells and to promote erythroblast production in humans, as it has an effect on leukemia cells in tro. , is being developed as a preventive and therapeutic agent for anemia caused by decreased red blood cell production (see JP-A No. 62-234097). [0009] However, it is not fully clear whether activin is a thrombopoietin-like physiologically active substance that acts on the megakaryocyte-platelet system, and it is even more difficult to measure the activin concentration in vivo. There have been no reports disclosing that the method is useful for predicting or diagnosing an increase or decrease in megakaryocyte-platelet count and/or megakaryocyte-platelet dysfunction, or for predicting or diagnosing diseases accompanied by these. [0010]An object of the present invention is to detect activin and/or activin-like substances in a target sample, which comprises using an antibody that recognizes at least a part of activin and/or activin-like substances. The objective is to provide a method for measuring [Means for Solving the Problems] The present inventors have previously discovered that activin and/or activin-like substances are used in megakaryocytes.
It has been found that it promotes differentiation, maturation and/or proliferation of platelet system (Japanese Patent Application No. 2-272866), and further,
We have been conducting intensive research on the relationship between activin and the megakaryocyte-platelet system. As a result, the measurement of activin and/or activin-like substances in a sample by a method comprising the step of using an antibody that recognizes activin and/or activin-like substances may result in an increase or decrease in the megakaryocyte-platelet axis and/or a decrease in the megakaryocyte-platelet axis. - We have completed the present invention by discovering that the present invention is useful for predicting and diagnosing functional abnormalities of platelet cells and diseases associated with these. [0012] That is, the present invention provides activin and/or
Alternatively, it is a measurement method characterized by measuring activin and/or an activin-like substance in a target sample, including a step of using an antibody that recognizes at least a part of the activin-like substance. The present invention will be explained in detail below. In the present invention, activin refers to two molecular species known to exist naturally and one molecular species predicted to exist naturally. That is, it is currently known that activin (activin A), which consists of a homodimer of βA chain, and activin (activin AB), which consists of a heterodimer of βA chain and βB chain, exist in nature. It is predicted that activin (activin B), which is composed of a homodimer of Alternatively, it may be a mixture of two or more of these. [0014] In the present invention, an activin-like substance is defined as having an amino acid sequence common to at least a part of any of the above-mentioned activins, and having at least an effect on the differentiation, maturation and/or development of the megakaryocyte-platelet system. Or it is a substance that has the activity of promoting proliferation. That is, any of the above-mentioned activins undergoes limited degradation by proteolytic enzymes, etc., resulting in a fragment, or the addition or substitution of one or more amino acids to the amino acid sequence of any of the above-mentioned activins.
This includes substances in which deletions have occurred. The activin-like substance targeted by the measurement method of the present invention may be any activin-like substance among the above-mentioned activin-like substances, or may be a mixture of two or more types. Furthermore, it may be a mixture of activin and an activin-like substance. [0015] The method for measuring activin and/or
Alternatively, the method includes a step of using an antibody that recognizes at least a part of the activin-like substance, and this antibody will be described below. The antibody used in the present invention may be any antibody that can recognize the above-mentioned activin and/or activin-like substance as an antigen. That is, it may recognize only one type of activin and/or activin-like substances described above, or it may recognize two or more types. Of course, it may be one that recognizes activin and activin-like substances. However, antibodies that recognize at least a portion of activin A are preferred. [0017] Furthermore, the antibodies used in the present invention may be polyclonal antibodies or monoclonal antibodies, or F(ab')2, Fab ' etc. [0018] The antibody used in the present invention is a B cell mRNA.
Antibodies may be produced by any method, such as genetic engineering using antibodies, immunization of animals, etc., but the following methods are common as methods for producing antibodies. First, an animal is immunized using activin and/or an activin-like substance, or a portion thereof, as an immunogen. [0020] The immunogen used here may be one that can obtain the desired antibody that recognizes the above-mentioned activin and/or activin-like substance, and may even be the entire activin and/or activin-like substance. It may be obtained from nature or may be artificially produced. [0021] When obtaining activin or an activin-like substance used as an immunogen from nature, human or animal tissues capable of producing activin and/or activin-like substances, such as placenta or ovaries, or human or animal urine or body fluids are used. from, known methods (e.g. Wylie Vale et al.
Extraction and purification may be carried out by referring to the above), or by culturing appropriate human cells or animal cells under appropriate conditions and purification from the culture supernatant. [0022] As cells that produce activin, leukocyte-derived cells such as THP-1 strain and U937 strain are known, so activin can be obtained by culturing these cells by a known method and then using a known purification method. (For example, Yuzuru
Eto etal. , Biochem. Biop
hys. Res. Commun. , Vol. 19
2, p. p. 1095-1103, 1986), it can be purified from the culture supernatant. [0023] Other cells that produce activin and/or activin-like substances include, for example, KHM-5M, a human-derived cell line. The present inventors developed the human-derived cell line KHM-5M in August 1990.
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM P-
11654) ). [0024] To artificially produce activin or an activin-like substance, a DNA encoding the amino acid sequence of the desired activin or activin-like substance is produced;
Appropriate host cells may be transformed with the DNA, and activin or activin-like substances may be recovered from the culture supernatant of the transformed host cells. This series of recombinant DNA manipulations can be carried out using known methods (for example, Maniatis T. e.
tal. , Molecular Cloning,
Cold Spring Harbor Labor
(1982)), and in particular, when activin A is obtained using recombinant DNA technology, the method of Masahiro Murata et al. (already published) may be referred to. It is also possible to chemically synthesize desired activin and/or activin-like substances. For chemical synthesis, for example, a peptide synthesizer 431A (manufactured by Applied Biosystems) may be used. As the immunogen for producing antibodies used in the present invention, activin A or a part of activin A is particularly preferable, and follicle-stimulating hormone secretion promoting activity,
Proliferation inhibitory activity and differentiation induction promoting activity against mouse friend leukemia cells and human erythroblastic leukemia cell line K-562, or differentiation, maturation and/or differentiation of megakaryocyte-platelet lineage.
Activin or activin-like substances having any of the following activities are also preferred. [0027]Currently, the following methods are known as methods for measuring each activity. Methods for measuring the secretion-promoting activity of follicle-stimulating hormone include the method of Wylie Vale et al. (previously published), the differentiation-promoting activity against Mouse Friend leukemia cells, and the growth-suppressing activity against the human erythroblastic leukemia cell line K-562. And as a method for measuring differentiation-promoting activity, Yu
The method of zuru Eto et al. (Biochem.
phys. Res. Commun. Vol. 1
42, p. p. 1095-1103, 1986) and the method of Yoshiyuki Miyamoto et al.
Biochem. Biophys. Res. Co
mmun. Vol. 168, p. p. 1149-1
156, 1990) are known. Furthermore, it is convenient to use the established mouse megakaryocyte cell line L8057 to measure the activity for differentiation, maturation, and/or proliferation of the megakaryocyte-platelet system (Journal of the Japanese Society of Hematology, Vol. 51, p. 310). , presentation number 187, 1988)
. Like mouse megakaryocyte cells, these cells produce acetylcholinesterase (AchE), an enzyme unique to megakaryocytes, as they mature.
The number of cells producing chE is measured, or the amount of AchE produced is measured spectrophotometrically (e.g. To
Shiro Nagasawa et al., Journal of the Japanese Society of Hematology,
49, pp. 1688-1695, 1986), it is possible to measure the effects of physiologically active substances on differentiation, maturation, and proliferation of megakaryocyte-platelet system. Activin and/or obtained as described above
Animals useful for immunizing animals using activin-like substances as immunogens are warm-blooded animals such as rabbits, sheep, pigs, mice, rats, guinea pigs, chickens, and goats. Moreover, the method of immunizing these animals may be any commonly used method. [0030] Next, antibodies are obtained from the immunized animal, and this method may also be performed by any conventionally known method. An example is as follows. When the antibody of interest is a polyclonal antibody, the antiserum obtained from the immunized animal is precipitated with 33% saturated ammonium sulfate to obtain an immunoglobulin G fraction (IgG fraction).
Separate. Next, it is determined whether this fraction has antiactivin and/or antiactivin-like substance activity. As a specific method for determination, an antibody is bound to the antigen adsorbed on a plate, and an enzyme-labeled secondary antibody is reacted with the antibody, according to a known enzyme-labeled immunosorbent assay (hereinafter abbreviated as ELISA method). Examples include a method of measuring enzyme activity by a coloring reaction of a substrate due to the action of a chemical, or a known radioimmunoassay method using an antigen labeled with a radioisotope, but various known methods can be used. be. [0032] When preparing a monoclonal antibody, the spleen cells of the immunized animal are fused with a parent cell for cell fusion such as a myeloma cell line, and a suitable one is selected from the resulting fused cells (hybridoma). Select and clone the fused cells, then in vitro
iro) or in vivo), and a highly specific monoclonal antibody is collected from this culture mixture. [0033] The antibody obtained as described above is purified and used if necessary. The purification method may be a combination of known methods such as salting out, dialysis, ion exchange chromatography, and affinity chromatography. [0034] The measurement method of the present invention includes a step of using the antibody obtained as described above, and this step involves combining activin and/or an activin-like substance, which is a test substance in a target sample, with the antibody obtained as described above. Preferably, this is a step of trapping the test substance in the target sample through an antigen-antibody reaction with the antibody obtained in the step. [0035] The principle of detection of the analyte in the measurement method of the present invention is not particularly limited, but examples include the agglutination method, the sandwich method, the solid phase direct method, and the competitive method. In the agglutination method, antibodies are bound to the surface of particles, such as latex particles or red blood cells (such as sheep red blood cells), such that agglutination of the particles occurs in the presence of activin and/or activin-like substances; Activin and/or activin-like substances are measured using the degree of activin as an indicator. In addition to latex and red blood cells, commonly used particles such as gelatin, microbeads, and carbon particles can be used in this agglutination method. In addition, in the sandwich method, solid phase direct method, and competitive method, labeled antibodies and antigens are used, and enzyme immunoassay (EIA) and radioimmunoassay (RI
A) The measurement can be performed based on the principles of chemiluminescence immunoassay (chemiluminescence immunoassay), fluoroimmunoassay (fluorescence immunoassay), time-resolved fluorescence immunoassay (TR-FIA), etc. The solid phase sandwich method based on the principle of EIA, which is one of the preferred examples of the measurement method of the present invention, will be described below.
Explain the solid phase direct method and solid phase competitive method. In the solid phase sandwich method using EIA, first, peroxidase, alkaline phosphatase, β
- Prepare an antibody that recognizes activin and/or an activin-like substance and is labeled with an enzyme such as galactosidase. Further, the solid phase used is adsorbed with an antibody that recognizes activin and/or an activin-like substance. After adding the sample or standard to the solid phase, the enzyme-labeled antibody described above is added to perform an antigen-antibody reaction. After removing excess enzyme-labeled antibody by washing,
A chromogenic substrate depending on the enzyme used, such as 1,2-phenylenediamine and H2O2, P-nitrophenyl phosphoric acid, 2-nitrophenyl-β-D-galactoside, etc., is added and reacted with the enzyme. Since the color development of a substrate depends on the amount of enzyme and ultimately on the amount of activin and/or activin-like substance in the sample, activin and/or activin-like substance can be quantified by measuring the amount of the colored final product. In the solid phase direct method, a sample is directly adsorbed onto a solid phase, and the surface of the solid phase on which activin and/or activin-like substances are not adsorbed is coated with proteins that do not affect the measurement system, such as BSA (bovine serum). After that, an enzyme-labeled antibody that recognizes activin and/or an activin-like substance is added and reacted. Thereafter, the same operation as in the solid phase sandwich method is performed to determine the presence or absence of activin and/or activin-like substance in the sample or to quantify it. In the solid phase competition method, a certain amount of activin and/or an activin-like substance recognized by the antibody used is directly adsorbed onto a solid phase, and then, after a blocking treatment,
Here, an enzyme-labeled antibody that recognizes activin and/or an activin-like substance and a sample are added. After reacting for a certain period of time, the solid phase is washed to remove unbound substances, and a chromogenic substrate is added to react with the enzyme. After the reaction, activin and/or activin-like substance in the sample is quantified by measuring the degree of inhibition of binding of the enzyme-labeled antibody to solid-phase activin and/or activin-like substance by adding the sample. [0043] First, the antibody is adsorbed on a solid phase, and enzyme-labeled activin and/or activin-like substance is added at the same time as the sample, and the degree of inhibition of the binding of the labeled substance to the immobilized antibody by the addition of the sample is measured. By this,
Activin and/or activin-like substances in the sample may be quantified. As a method other than the above, the antigen-antibody reaction is carried out in a liquid phase rather than a solid phase, and then activin and/or activin-like molecules bound to a labeled antibody are analyzed by coagulation sedimentation or physicochemical techniques using antibodies. There is also a method of quantifying the substance by making it insoluble. Additionally, activin and/or
Alternatively, instead of labeling an antibody that recognizes an activin-like substance, obtain a secondary antibody that recognizes that antibody, label it, and perform an antigen-antibody reaction to measure activin and/or an activin-like substance. is also possible. In any of the sandwich method, solid phase direct method, and competitive method, the combination of labeled enzyme and chromogenic substrate can be changed to combinations of labeled enzyme and bioluminescent substrate, chemiluminescent substrate, labeled enzyme and fluorescent substrate, etc. It is possible. In this case, typical enzyme-luminescent substrate combinations include alkaline phosphatase-AMPPD, horseradish peroxidase-luminol, luciferase-luciferin, etc., and typical enzyme-fluorescent substrate combinations include alkaline phosphatase-umbelliferyl. phosphate, horseradish peroxidase-p-hydroxyphenylpropionic acid, and the like. Furthermore, in the above three measurement methods, instead of enzymes, antibodies or antigens labeled directly or indirectly with radioactive substances, chemiluminescent substances, or fluorescent substances are used to detect radioactivity, luminescence, or fluorescence. By measuring the intensity it is also possible to determine activin and/or activin-like substances in the sample. [0047] Radioactive substances include 125I and 13
1I and the like are generally used, and typical chemiluminescent substances include acridinium ester and the like. In addition, when measuring fluorescence intensity, a more sensitive method is
activin and/or Methods for measuring activin-like substances (time-resolved immunofluorescence assay) are also useful. Note that europium is an example of a typical rare earth metal. As explained above, the measuring method of the present invention is aimed at measuring activin and/or activin-like substances in a sample. In this case, the target sample is not limited, but is preferably a body fluid. More preferably, the sample is selected from blood, plasma, serum, urine, spinal fluid, lymph, saliva, ascites, and pleural fluid. The measurement method of the present invention described above can be used to detect increases or decreases in the megakaryocyte-platelet system and/or abnormalities in megakaryocyte-platelet function and diseases associated therewith, such as bone marrow tumors and aplastic anemia. Useful as a prognostic and diagnostic aid. [0050] The measurement method of the present invention can also be used during the use of drugs that have a large effect on the megakaryocyte-platelet system, such as drugs such as anticancer drugs and antibiotics that are known to cause a decrease in platelet count as a side effect, or during radiotherapy. Provide information for determining dosage and usage in cases such as [Examples] The present invention will be explained in more detail with reference to Examples below, but these are shown as examples of implementation, and the present invention is not limited by these in any way. The abbreviations used in the following description are based on common abbreviations in the field, and techniques related to genetic recombination are referred to in "Mania" unless otherwise specified.
tis, T. et al. , Molecular
Cloning, Cold Spring Harbo
The experiments were carried out in accordance with the protocols attached to the reagents and instruments as well as books such as "Practical Handbook of Genetic Manipulation Experiments, Translated by Shigeyasu Kobayashi (Jatech Publishing Co., Ltd. (1985)")" (Example 1) Production of activin using THP-1 cell line (1) Culture of THP-1 cells The THP-1 cell line was treated with 5% FCS (fetal bovine serum, manufactured by Irvine Scientific). /RPMI16
40 (manufactured by Gibco), 1 x 105 cells/ml
3 liters of cell suspension was obtained. This was placed in a 20 liter roller bottle (manufactured by Suzuki Seiko Co., Ltd.) and cultured at 30 rotations/hour at 37° C. for 4 days. After culturing, the cells are collected by centrifugation, and the collected cells are transferred to RPMI1640.
After washing three times with 1 liter of medium, 100 ng/ml PMA (
4β-phorbol-12-myristate-1
RPMI1 containing 3-acetate (manufactured by Sigma)
The cells were inoculated into 3 liters of 640 medium at a density of 5 x 105 cells/ml, and cultured in a 20 liter roller bottle at 30 rotations/hour at 37°C for an additional 4 days. After the culture was completed, the cell suspension was collected and centrifuged to obtain a culture supernatant. (2) Purification of activin from THP-1 cell culture supernatant The obtained culture supernatant was purified with a molecular weight cutoff of 1000.
It was concentrated at 4° C. using a 0.0 ultrafiltration membrane (Pericone Polysulfone Membrane, manufactured by Millipore). After concentration, 70
Ammonium sulfate was added to perform salting out to achieve % saturation, followed by centrifugation at 10,000 xg at 4°C for 30 minutes. The precipitate was collected, dissolved in distilled water, and thoroughly dialyzed against 0.05M Tris-HCl buffer (pH 7.8) at 4°C. The sample after dialysis was purified by anion exchange chromatography using the following method. [0054] That is, the sample after dialysis was added to a DEAE-Toyopearl 650M column (10.8 cmφ x 20 cm, manufactured by Tosoh Corporation) that had been equilibrated in advance with 0.05 M Tris-HCl buffer (pH 7.8). did. After washing with 0.05M Tris-HCl buffer (pH 7.8), the amount of protein in the eluate fraction was monitored by measuring the absorbance at a wavelength of 280 nm.
M NaCl/0.05M Tris-HCl buffer (pH 7
．． 8), elution was performed by the concentration gradient method. Collect a portion from each fraction and add BS to a final concentration of 0.1%.
After adding A, the mixture was dialyzed against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile filtration membrane (Millex GV filter with a pore size of 0.22 μm, manufactured by Millipore), and the
．． It was diluted with 1% BSA/RPMI1640 and used as a sample for activity measurement. When the activity of the obtained sample was measured by the method described in Example 4, activity was observed in the fraction with a NaCl concentration of about 0.1M. [0055] Ammonium sulfate was added to the obtained active fraction to achieve 70% saturation for salting out. 100
Centrifugation was performed at 00xg for 10 minutes at 4°C to collect the precipitate, which was dissolved in distilled water. After dissolution, an equal volume of 80% saturated ammonium sulfate solution was added, and the mixture was purified by hydrophobic chromatography using the following method. First, Butyl Toyopearl 650M column (
2.5cmφ×30cm, manufactured by Tosoh Corporation), 40
% saturated ammonium sulfate (pH 7.8), and the above sample was added thereto. 40% saturated ammonium sulfate/0.05M
After washing with Tris-HCl buffer (pH 7.8), elute with 20% saturated ammonium sulfate/0.05M Tris-HCl buffer (pH 7.8) and then 20% ethanol while monitoring the protein amount in the elution fraction. I did it. A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, perform sterilization filtration using a sterile Mirex GV filter (previously listed), dilute with 0.1% BSA/RPMI1640,
This was used as a sample for activity measurement. The activity of the obtained sample was measured by the method described in Example 4, and the activity was as follows.
It was observed in the 20% ethanol elution fraction. The obtained active fractions were collected, ethanol was removed by vacuum concentration, and then a sufficient amount of 0.05M Tris-HCl buffer (pH 8.0) was added using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000.
dialysis was performed thoroughly at 4°C. The sample after dialysis was purified by anion exchange chromatography using the following method. [0057] That is, the above sample was prepared in advance at a temperature of 0.05
Mo equilibrated with M Tris-HCl buffer (pH 8.0)
noQ HR5/5 column (0.5cmφ×5cm,
(manufactured by Pharmacia) and washed with the same buffer. After washing, while monitoring the protein amount in the elution fraction, 0M-0.
2M NaCl/0.05M Tris-HCl buffer (pH
Elution was performed by a concentration gradient method using 8.0). A portion of each fraction was collected and B was adjusted to a final concentration of 0.1%.
After adding SA, the mixture was dialyzed against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 0.1% BSA/RPMI1640 to prepare a sample for activity measurement. When the activity of the obtained sample was measured by the method described in Example 4,
Activity was observed at a NaCl concentration of approximately 0.1M. After adding trifluoroacetic acid to the obtained active fraction to a final concentration of 0.1%, it was subjected to reverse phase chromatography.
It was purified by the following method. That is, a Hypore RP304 column (0.46c) equilibrated with 0.1% trifluoroacetic acid in advance
The above sample was added to a tube (mφ×25 cm, manufactured by Bio-Rad) and washed with 0.1% trifluoroacetic acid. Next, while monitoring the protein amount in each fraction,
Elution was performed using n-propanol/0.1% trifluoroacetic acid using a linear concentration gradient method. Collect a portion from each fraction, add BSA to a final concentration of 0.1%, and then use a dialysis membrane with a molecular weight cutoff of 6000 to 8000.
Dialyzed against PBS-. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 0.1% BSA/RPMI1640 to prepare a sample for activity measurement. The activity of the obtained sample is
When measured by the method described in Example 4, activity was observed in fractions with n-propanol concentrations around 25%. (3) Amino acid sequence determination Example 1 (2)
After freeze-drying the active fraction finally obtained by the method, a part of it was dissolved in 50% acetic acid to prepare a sample, and a model 47
7A Protein Sequencing System-120AP
The amino acid sequence was determined using a TH amino acid analyzer (manufactured by Applied Biosystems). The PTH amino acid was detected by ultraviolet absorption at 270 nm, and the amino acid was identified based on the retention time of a standard PTH amino acid (manufactured by Applied Biosystems) that had been previously separated using the same method. As a result, this protein was confirmed to have the amino acid sequence shown in SEQ ID NO:1. SEQ ID NO: 1: Gly Leu Glu Xaa Asp Gly L
ys Val AsnIle Xaa Xaa Lys
Lys Gln Phe Phe Val Xaa
Phe (Xaa indicates that the amino acid is not specified).The remaining active fraction (lyophilized product) finally obtained was used for subsequent experiments. (Example 2) Human-derived cell line KHM-5M
Production of activin and/or activin-like substances using (1) Culture of human-derived cell line KHM-5M in 5% FBS (
Inactivated fetal bovine serum (manufactured by Sera Labo)/RPMI16
Approximately 2 x 10 human-derived cell line KHM-5M in 40 medium
The cells were implanted at a concentration of 4 cells/ml and cultured at 37° C. for 3 days at 30 rotations/hour in a 5 liter roller bottle (manufactured by Tanehashi Kikaiten Co., Ltd.). After culturing, the medium was removed and washed 3 times with 250 ml of RPMI1640 medium, then replaced with 1 liter of IMDM (Iscove's MDM, manufactured by Gibco) medium, and incubated at 30 rpm for another 3 days.
The cells were cultured at 37°C for an hour. After the culture was completed, the cell suspension was collected and centrifuged at 8000 xg and 4°C for 20 minutes to obtain a culture supernatant. (2) Purification of activin and/or activin-like substance from human-derived cell line KHM-5M culture supernatant [0062] The obtained culture supernatant was filtered using an ultrafiltration membrane (Spiral Cartridge S1Y3, manufactured by Amicon). ) and further concentrated using an ultrafiltration membrane (YM5, manufactured by Amicon). The sample after this concentration has a molecular weight cutoff of 350.
50mM sodium acetate buffer (p
Dialysis was performed overnight at 4° C. against H5.0). The sample after dialysis was centrifuged at approximately 90,000 xg and 4°C for 20 minutes, and the supernatant was collected. The collected supernatant was purified by cation exchange chromatography using the following method. That is, SP-Toyopearl 6 equilibrated in advance with 50mM sodium acetate buffer (pH 5.0)
The sample after dialysis was added to a 50S column (2.2 cmφ×20 cm, manufactured by Tosoh Corporation). After washing with the same buffer, elution was performed using a 0M to 1.0M NaCl/50mM sodium acetate buffer (pH 5.0) using a linear concentration gradient method. A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed against PBS- overnight using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration is performed using a sterile Mirex GV filter (previously published), and 0.1
%BSA/IMDM to prepare a sample for activity measurement. The activity of the obtained sample was measured by the method described in Example 5, and the activity was found to be approximately 0.15 at NaCl concentration
It was observed in the fraction around M~0.35M. The active fraction was collected and further purified by anion exchange chromatography using the following method. That is, the above active fraction was converted into YM5 (already mentioned).
The concentrated sample obtained was diluted with 25mM Tris-HCl buffer (
(pH 8.0) for 3 hours. The sample after dialysis was centrifuged at approximately 90,000 xg and 4°C for 20 minutes, and the supernatant was collected. This supernatant was applied to a DEAE-5PW column (7.5 mmφ x 75 mm,
(manufactured by Tosoh Corporation). After washing with the same buffer,
0M-1.0M while monitoring the protein amount in the elution fraction by measuring absorbance at a wavelength of 280 nm.
NaCl/25mM Tris-HCl buffer (pH 8.0
) Elution was performed by linear concentration gradient method. Collect a portion from each fraction and add BSA to a final concentration of 0.1%.
was added, and then dialyzed against PBS- overnight using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, perform sterilization filtration using a sterile Mirex GV filter (previously listed), dilute with 0.1% BSA/IMDM,
This was used as a sample for activity measurement. The activity of the obtained sample was measured by the method described in Example 5, and it was found that NaCl
Two peaks of activity were observed in fractions around concentrations of about 0.05M to 0.35M. The elution pattern is shown in Figure 1. In the figure, a fraction corresponding to peak B was collected and purified by reverse phase chromatography using the following method. That is, the above active fraction was converted into YM5 (already mentioned).
The concentrated sample obtained was concentrated using a dialysis membrane with a molecular weight cutoff of 8000, and 0.1% trifluoroacetic acid/
Dialysis was performed against 10% acetonitrile for 3 hours. For the sample after dialysis, approximately 90,000 x g, 4°C, 2
Centrifugation was performed for 0 minutes, and the supernatant was collected. This supernatant was applied to a PLRP-S column (4.6 mmφ) equilibrated in advance with 0.1% trifluoroacetic acid/10% acetonitrile.
×50 mm, manufactured by Polymer Laboratories, Inc.). After washing with 0.1% trifluoroacetic acid/10% acetonitrile, the amount of protein in the eluted fraction was monitored by measuring the absorbance at a wavelength of 280 nm.
Elution was performed using a linear concentration gradient method with 1% trifluoroacetic acid/10%-90% acetonitrile. A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 0.1% BSA/IMDM to prepare a sample for activity measurement. The activity of the obtained sample was measured according to the method described in Example 5. As shown in Table 1, the fraction with acetonitrile concentration of about 30% was
It was found to have an effect on the megakaryocyte-platelet system. (3) Amino acid sequence determination Example 2 (2)
After freeze-drying the active fraction finally obtained, a portion thereof was used to analyze the amino acid sequence by the method described in Example 1 (3). As a result, it was confirmed that the activin or activin-like substance in the active fraction had the amino acid sequence shown in SEQ ID NO:2. SEQ ID NO: 2: Gly Leu Glu Xaa Asp Gly L
ys Val AsnIleXaa Xaa Lys
Lys Gln Phe Phe Val XaaPh
eLys Asp Ile Gly (Xaa indicates that the amino acid is not specified.) The remainder of the finally obtained active fraction (lyophilized product) was used for subsequent experiments. (Example 3) Production of activin A by recombinant DNA technology (1) Obtaining cDNA encoding activin Polymerase Chain Reacti by the following method
on (PCR method) to amplify and clone cDNA encoding activin. First, PCR
sense primer A and antisense primer A
The base sequence encoding activin A (Anth
one J. Mason et al. , Bioc
hem. Biophys. Res. Commun
．． , Vol. 135, p. p. 957-964
, 1986).
Each was produced using a chemical synthesizer (Model 381A, manufactured by Applied Biosystems) (see SEQ ID NO: 3, SEQ ID NO: 4, and FIG. 2). After synthesis, each was purified using an OPC column (manufactured by Applied Biosystems), and then using T4 polynucleotide kinase (manufactured by Takara Shuzo).
The 5' end was phosphorylated. Next, these were purified using Sephadex G-25 (manufactured by Pharmacia) and used as sense primer A and antisense primer A in subsequent operations. On the other hand, total RNA was extracted using the guanidine thiocyanate method using about 20 g of human placenta as a material. About 90 μg of polyA+ RNA was purified by passing 10 mg of the obtained RNA twice through an oligo dT cellulose (type 7, manufactured by Pharmacia) column. Next, cDNA was cloned using this poly A+ RNA as a material. First, poly A+
Single-stranded cDNA was prepared from 2.5 μg of RNA using oligo dT as a primer (cDNA synthesis system (
(manufactured by Amersham). Next, for the obtained single-stranded cDNA,
PCR was performed using the sense primer A and antisense primer A as described below. That is, about 0.2 μg of cDNA, sense primer A0.
8 μg and antisense primer A0.8 μg,
PCR buffer (20mM Tris-HCl buffer (pH 8.
3)/1.5mM MgCl2 25mM KCl
/0.05% Tween20/100 μg/ml gelatin/200 μM dNTP) and add 2 units of Taq DNA polymerase to this solution.
(manufactured by Takara Shuzo Co., Ltd.) was added to cause a reaction. The reaction temperature and time were 94°C for 1 minute in the denaturation stage, 55°C for 2 minutes in the annealing stage, and 2 minutes at 55°C in the enzymatic reaction stage.
The temperature was 72°C for 3 minutes, and this cycle was repeated 30 times in total. After recovering the generated DNA by chloroform treatment and ethanol precipitation, it was further treated with T4 DNA polymer.
The resulting DNA was made blunt-ended using ase. After the reaction, the obtained DNA was subjected to 0.8% agarose gel electrophoresis, and the generated DNA fragment of approximately 1.3 kb was extracted from the gel, and the desired DNA was purified by phenol treatment, chloroform treatment, and ethanol precipitation. , recovered. (2) Expression and purification of activin The DNA obtained in Example 3 (1) was cloned into the mammalian expression vector pCDM8 by the following method. That is, a BstXI linker was added to the 1.3 kb DNA with T4DN.
After connecting with A ligase, gel filtration (Sephadex G-50, manufactured by Pharmacia) was performed to separate the desired DNA connected with the linker from the remaining linker. Next, the DNA was digested with BstXI and cloned into plasmid pCDM8 from which the stuffer DNA had been removed (the above procedure was performed using THE LIBRARIA
(Using NTMI (manufactured by Funakoshi Pharmaceutical Co., Ltd.)). E. coli strain MC1061/P3 was transformed with this plasmid pCDM8. Next, the plasmid DNA in the obtained tetracycline-resistant strain was analyzed to obtain a plasmid in which cDNA encoding activin was connected in the forward direction to the cytomegalovirus promoter in plasmid pCDM8, and this was transformed into an activin expression plasmid. I gave it an A. Various restriction enzyme-cleaved fragments of the activin-encoding DNA portion of this plasmid were digested with M13 phage m
Subcloned into p18 and mp19. After preparing single-stranded phage DNA from each, use a DNA sequencer (370A, manufactured by Applied Biosystems)
When the base sequence was determined, the desired DNA sequence was confirmed. [0073] The obtained activin expression plasmid A was
CHO lacking dihydrofolate reductase (DHFR)
DXB11 strain (Urlaub G. & Chas
in L. A. , Proc. Natl. Aca
d. Sci. USA. , Vol. 77, p.
p. 4216-4220, 1980: Hereinafter, CH
plasmid p encoding DHFR
AdD26SVpA (Kaufman R.J. &
Sharp P. A. , J. Mol. Biol
．． , vol. 159, p. p. 601-621
, 1982) by electroporation, and cell clones expressing activin were isolated. That is, 50 μg of activin expression plasmid A and 2.5 μg of pAdD26S were added to 7×106 semiconfluent CHO cells.
The mixed DNA containing VpA was transfected using an electroporation device (manufactured by DP Systems). After 2-4 days, the medium was replaced with a selective medium of MEMα(-) (Gibco) containing 10% dialyzed fetal bovine serum. The medium was exchanged at intervals of 2 to 4 days, and colonies produced in 3 to 4 weeks were isolated. After growing each of these isolated colonies, each was grown at 0.
Dulbecco's modified Eagle's medium containing 01% albumin (
The cells were cultured in DMEM (manufactured by Gibco) for 3 days, and a portion of the culture supernatant was collected. For each culture supernatant collected,
Example 4
Activity was measured by the method described in . From the obtained results, one clone that was considered to have high activity was selected, and after growing it again, the culture supernatant was collected, and activin was purified according to the method described in Example 1 (2). I did it. (3) Confirmation of amino acid sequence After lyophilizing the active fraction finally obtained through purification, a part of it was used,
The amino acid sequence was analyzed by the method described in Example 1 (3). As a result, this protein was confirmed to have the amino acid sequence shown in SEQ ID NO:5. SEQ ID NO: 5: Gly Leu Glu Xaa Asp Gly L
ys Val AsnIle (Xaa indicates that the amino acid is not specified.) The remainder of the finally obtained active fraction (lyophilized product) was used for subsequent experiments. (Example 4) Human erythroblastic leukemia cell line K
- Measurement of activin activity using 562 cells The following method was used to measure the activity of activin in the human erythroblastic leukemia cell line K-56.
The differentiation-promoting activity against No. 2 was measured using hemoglobin production as an index. First, 2mM L-glutamine, 25mM
M HEPES, 50IU/ml penicillin, 50μ
Prepare a RPMI1640 medium containing g/ml streptomycin and 10% FCS (previously described), and add approximately 2 x 104 human erythroblastic leukemia cell line K-562 cells to this medium.
It was suspended so that the amount of cells/ml was obtained. Next, this cell suspension was added to each well of a 96-well multiplate (manufactured by Becton Dickinson) at 0.0%. Add 1 ml at a time, and
0.01 ml of the sample was added to the experimental group, and 0.01 ml of RPMI1640 medium was added to the control group, and cultured at 37° C. for 5 days. After culturing, add 0.01ml of benzicine reaction solution to each well.
was added, and then a portion of each well was collected into a counting chamber, the number of stained positive cells was counted, and the number of stained positive cells per 200 cells was calculated. Note that 2% dianisidine/3% acetic acid/3% hydrogen peroxide was used as the benzicine reaction solution, and hydrogen peroxide was added immediately before use. Further, the differentiation promoting activity of the sample against the human erythroblastic leukemia cell line K-562 was determined to be active when the number of positive staining cells per 200 cells was significantly higher than that of the control. (Example 5) Measurement of the effect of activin and/or activin-like substances on the megakaryocyte-platelet system The effect of activin and/or activin-like substance on the megakaryocyte-platelet system was measured by the following method. Ta. First, mouse megakaryocytic cell line L8057 was incubated with 1% neutoridoma-SP (Boehringer).
The cells were prepared at 3 x 104 cells/ml using Mannheim Yamanouchi Co., Ltd./IMDM medium, and cultured at 37°C for 3 days. After the completion of the culture, centrifuge at 1000 °C in a low-speed centrifuge (Tomy RL-100, manufactured by Tomy Seiko Co., Ltd.)
Cells were collected by centrifugation at revs/min for 5 minutes. The collected cells were mixed with 2% nutoridoma-SP/
0.1% BSA (manufactured by Sera-Labo)/IMDM medium,
The cells were suspended at 3 x 104 cells/ml to obtain a cell suspension for activity measurement. Activity was measured by the following method. First, a 96-well multi-plate was prepared, and 100 μl/well of the above sample was added to each well for the experimental group and 0.1% BSA/IMDM for the control group. Next, 100 μl of the above cell suspension for activity measurement was added to each well and cultured at 37° C. for 3 days. After culturing, the plate was centrifuged for 5 minutes at 1500 rpm using a centrifuge (H-161, manufactured by Kokusan Centrifuge Co., Ltd.), and then 150 μl of the supernatant was removed from each well. Instead, 150 μl of PBS adjusted to pH 7.2 was added. Then 0
．． 315% DTNB/1% sodium citrate to 1% T
Diluted 10 times with riton
tiskan MCC), the absorbance at a wavelength of 405 nm was measured. After measuring the absorbance, 20 μl of a 7.5 mM acetylthiocholine iodide solution was added to each well, and after reacting at 25° C. for 30 minutes, the absorbance at a wavelength of 405 nm was measured again. Then, the difference between the two measured absorbances, ie, ΔOD405 (30 min −0 min), was calculated. Note that DTNB is 5,5'-dithiobi
It is an abbreviation for s-(2-nitrobenzoic acid). In addition, as a blank for absorbance measurement, P
BS- was used. When a significant difference from the control was observed, it was determined that there was an effect on the megakaryocyte-platelet system. (Example 6) Preparation of anti-activin polyclonal antibody and labeled anti-activin polyclonal antibody (Part 1) (1) Preparation of anti-activin polyclonal antibody Example 1
0.5ml of 0.1mg of activin finally obtained in
FCA (Freund's
complete adjuvant) 0.5m
1 to prepare an emulsion, which was inoculated intradermally into a male New Zealand White Rabbit (2.5 kg, Charles River Japan Co., Ltd.). Then activin 0
．． Dissolve 1 mg in 0.5 ml of physiological saline and add it to F
IA (Freund's incomplete ad)
juvant) to prepare an emulsion and administer it subcutaneously 2 weeks after the first vaccination.
Administered 1 to 3 times weekly. Two weeks after the final administration, whole blood was collected to obtain antiserum. 6 ml of rabbit antiserum thus obtained
Add ammonium sulfate to 40% saturation, precipitate the solution, centrifuge, dissolve the resulting precipitate in 3 ml of 5 mM phosphate buffer (pH 7.2), and add ammonium sulfate to 40% saturation.
Dialysis was performed for 24 hours against H7.2). The sample after dialysis
DEA equilibrated with 5mM phosphate buffer (pH 7.2)
Apply it to an E cellulose column, separate the non-adsorbed fraction, and
Dialyzed against S- for 24 hours. As a result, 3.6
A mg/ml IgG solution was obtained. (2) Preparation of labeled anti-activin polyclonal antibody 5 mg of horseradish peroxidase was added to PBS-
IgG obtained in Example 6 (1) dissolved in 1 ml
After dialysis against PBS- at 4°C, 25% glutaraldehyde was added dropwise to a final concentration of 0.2% and mixed. This was dialyzed against PBS- at 4°C to obtain a peroxidase-labeled polyclonal antibody. (Example 7) Preparation of anti-activin-like substance polyclonal antibody and labeled anti-activin-like substance polyclonal antibody (1) Preparation of anti-activin-like substance polyclonal antibody Polyclonal antibody against the activin-like substance finally obtained in Example 2 Antibodies were produced in the same manner as in Example 6(1). (2) Preparation of labeled anti-activin-like substance polyclonal antibody The rabbit anti-activin-like substance polyclonal antibody obtained in Example 7 (1) was labeled with peroxidase in the same manner as in Example 6 (2). Ta. (Example 8) Preparation of anti-activin polyclonal antibody and labeled anti-activin polyclonal antibody (Part 2) (1) Preparation of anti-activin polyclonal antibody Example 3
A polyclonal antibody against the recombinant activin obtained in Example 6(1) was produced in the same manner as in Example 6(1). (2) Preparation of labeled anti-activin polyclonal antibody The rabbit anti-activin IgG solution obtained in Example 8 (1) was labeled with peroxidase in the same manner as in Example 6 (2). (Example 9) Preparation of anti-activin monoclonal antibody and labeled anti-activin monoclonal antibody (Part 1) (1) Preparation of anti-activin monoclonal antibody Example 1
The activin finally obtained was dissolved in physiological saline,
This was mixed with FCA in equal amounts to create an emulsion, and B
ALB/c mice (male, 4 weeks old, Japan Charles River Co., Ltd.) were immunized by subcutaneously administering 30 μg/mouse of activin three times at one week intervals. . One week later, a final immunization was performed by intravenously injecting only 5 μg of activin solution. Four days after the final immunization, the mice were sacrificed and the spleens were removed. After this was shredded, it was passed through a stainless steel mesh and suspended in MEM medium to obtain a splenocyte suspension. The splenocytes and mouse myeloma cells (P3U1 cells) were each washed three times with MEM,
Splenocytes and myeloma cells were mixed at a ratio of 4:1 and centrifuged (1400 rpm, 8 minutes). In the obtained precipitate,
1 ml of 40% polyethylene glycol 4000/10% dimethyl sulfoxide/10 μg/mg poly-L-arginine/PBS-solution was gradually added and left at room temperature for 1 minute to perform cell fusion. Next, 2ml of MEM
was added for 2 minutes, then 1 ml of MEM was added for 3 minutes, and finally the total volume was made up to 10 ml with MEM, and then centrifuged (800
rpm for 5 minutes). This precipitate was mixed with 10% FC.
The cells were suspended in S/RPMI1640 medium at 2×10 5 cells/ml and dispensed into a 96-well microplate in an amount of 0.2 ml per well. The next day, HAT (hypoxanthine 1 x 10-4M, aminopterin 4 x 10-7M,
10% FCS/ containing thymidine (1.6 x 10-5M)
The medium was replaced with RPMI1640 (HAT medium). Thereafter, when half of the culture medium was replaced with HAT medium every 3 to 4 days, colonies of hybridoma cells were observed from around the 7th day. In order to examine the presence or absence of antibodies against activin in the culture supernatant of hybridoma cells, ELI was performed using a 96-well ELISA plate to which activin was adsorbed.
Screening was performed using the SA method. The antigen used in the screening was the activin obtained in Example 1, and the second antibody was alkaline phosphatase-labeled sheep anti-mouse IgG. When the number of cells in each well that were positive by ELISA increased to approximately 104
T medium was added. The medium was replaced with HT medium twice every 3 to 4 days, and then the usual 10% FCS/
Culture was performed using RPMI1640 medium. When the number of cells in each positive well has increased sufficiently, the hybridoma cells are
2 pieces per well of a 96-well microplate, 1 piece,
Or 10%FCS/RPMI1 to make 0.5 pieces
640, BALB/c mouse thymocytes were added as feeder cells, and the cells were implanted into each well of the plate and cultured. Observe under a microscope to select wells that are sure to contain single cell colonies, and screen those culture supernatants by ELISA to check for the production of anti-activin antibodies, and produce anti-activin monoclonal antibodies. I got a hybridoma. Next, in order to produce a large amount of monoclonal antibodies against activin, approximately 10 7 hybridoma cells producing anti-activin antibodies were intraperitoneally injected into BALB/c mice that had been pretreated with pristane. did. The ascites fluid collected on the 9th day was centrifuged, the supernatant was filtered through glass wool, and purified by affinity chromatography using protein A. After purification, dialysis was performed against PBS- at 4°C. The class of the monoclonal antibodies thus obtained was determined by an Ouchterlony gel diffusion test using class-specific anti-mouse antisera. As a result, the class of the monoclonal antibody obtained was IgG. (2) Preparation of labeled anti-activin monoclonal antibody Using the monoclonal antibody obtained in Example 9 (1), a peroxidase-labeled anti-activin monoclonal antibody was obtained in the same manner as in Example 6 (2). (Example 10) Preparation of anti-activin-like substance monoclonal antibody and labeled anti-activin-like substance monoclonal antibody (1) Preparation of anti-activin-like substance monoclonal antibody Using the activin-like substance finally obtained in Example 2 hand,
A monoclonal antibody against an activin-like substance was obtained in the same manner as in Example 9 (1). Note that the class of the obtained antibody was IgG. (2) Preparation of labeled anti-activin-like substance monoclonal antibody Using the monoclonal antibody obtained in Example 10 (1), peroxidase-labeled anti-activin-like substance monoclonal antibody was prepared by the method according to Example 6 (2). Obtained. (Example 11) Preparation of anti-activin monoclonal antibody and labeled anti-activin monoclonal antibody (Part 2) (1) Preparation of anti-activin monoclonal antibody Example 3
Using the activin finally obtained in Example 9 (1
) A monoclonal antibody against activin was obtained in the same manner. Note that the class of the obtained antibody was IgG. (2) Preparation of labeled anti-activin monoclonal antibody Using the monoclonal antibody obtained in Example 11 (1), a peroxidase-labeled anti-activin monoclonal antibody was obtained in the same manner as in Example 6 (2). (Example 12) Measurement using a solid phase sandwich method based on the principle of ELISA An activin-like substance measurement system using a solid phase sandwich method based on the principle of ELISA was prepared as follows. Example 7 (1) was added to a 96-well ELISA plate (NUNC Immunoplate II).
The polyclonal antibody obtained in 10μg/
Add 50 μl of the diluted solution to each well and let it stand at room temperature for 2 hours to allow it to adsorb onto the plate surface.
Each well was washed five times with PBS containing 0.001% Tween20. As a sample, the activin-like substance finally obtained in Example 2 was mixed with 1% BSA/0.001% T.
Ween20/PBS- (hereinafter referred to as EIA buffer) diluted to various concentrations were used, and 50 μl of each was added to the wells and allowed to stand for 1 hour. 0.001%
After washing five times with PBS containing Tween 20, 50 μl of the peroxidase-labeled polyclonal antibody obtained in Example 7 (2) diluted with EIA buffer to a concentration of 6 μg/ml was added to each well. Let it stand for a while. This was added to PBS containing 0.001% Tween20.
Wash 5 times with peroxidase chromogenic substrate (1,2-
Phenyldiamine 3mg/ml/0.027%H2
02, phosphate-citrate buffer, pH 5.0) was added to each well. After 30 minutes, the reaction was stopped by adding 50μl of 4N sulfuric acid to each well, and 490n
The absorbance of m was measured using Immunoreader NJ2000 (manufactured by Nippon Intermed Co., Ltd.). As a result, there was a positive correlation between absorbance and activin-like substance concentration, and almost linearity was obtained between activin-like substance concentrations of 0.01 to 1.0 μg/ml. (Example 13) Measurement using a solid phase competition method based on the principle of ELISA An activin measurement system using a solid phase competition method based on the principle of ELISA was prepared as follows. The activin obtained in Example 3 was prepared in PBS to a concentration of 4 μg/ml, and 50 μl of this was added to each well of a 96-well ELISA plate and left to stand for 2 hours to allow activin to be adsorbed to the plate surface. . Fill wells with 0.001% Tween2
After washing five times with PBS-0, 1 μg of the peroxidase-labeled polyclonal antibody obtained in Example 6 (2) was added.
The activin finally obtained in Example 1 was used as an EIA buffer and a sample at a concentration of /ml.
50 μl of each diluted with IA buffer was added to each well, and after 1 hour, 0.001%
Washed 5 times with PBS containing Tween 20 and prepared in Example 1.
Add 100 μl of the peroxidase chromogenic substrate shown in 2.
The reaction was stopped by adding μl at a time, and the absorbance at 490 nm was measured using Immunoreader NJ2000 (previously published). The concentration of activin in the test liquid is 3 to 0.03 μg
There was a correlation between absorbance and activin concentration between 100% and 100% activin concentration. (Example 14) Measurement using a solid phase direct method based on the principle of ELISA An activin measurement system using a solid phase direct method based on the principle of ELISA was prepared as follows. As samples, SEQ ID NOs: 6 and 7 (see Figure 3,
In the figure, recombinant activin AB having the amino acid sequence shown in (a) is the βA chain and (b) is the βB chain) was
Diluted with S- to various concentrations, 96-well ELISA
50 μl per well was placed in a plate for use, and the mixture was allowed to stand at room temperature for 2 hours to be adsorbed onto the plate surface. 0.001%Tw
Each well was washed five times with a PBS solution containing een20, and then 50 μl of an EIA buffer containing the perkiosidase-labeled polyclonal antibody obtained in Example 7 (2) at a concentration of 5 μg/ml was added to each well. . 1 at room temperature
After standing for a period of time, PBS- containing 0.001% Tween20
After washing with the solution, 100 μl of the same peroxidase coloring substrate as in Example 12 was added to each well. After 30 minutes, 50 μl of 4N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm was measured using Immunoreader NJ20.
00 (already mentioned). The absorbance was correlated with the activin concentration, and almost linearity was obtained between activin concentrations of 0.03 and 1 μg/ml. Note that even when the activin-like substance obtained in Example 2 was used as a sample in the same measurement system, a correlation was observed between the absorbance and the concentration of the activin-like substance. (Example 15) Radioimmunoassay The method of Onoue et al. and Nishi et al. (respectively, "Immunological Experiment Procedure A")
The activin-like substance was labeled with 125 I according to 1978, pp. 174-175 and 189-195), edited by the Japanese Society of Immunology. That is, a 0.5 M phosphate buffer solution (pH 7.0) containing 2 μg of the activin-like substance finally obtained in Example 2 was placed in a siliconized test tube.
5) Add 5 μl of Na125 I (0.5 mCi) to 25 μl
Add 40 μl of chloramine T solution and incubate at room temperature for 1 hour.
The reaction was allowed to take place while stirring for a minute. 3mg/mlNa2S
After stopping the reaction by adding 20 μl of O3, add 0.1% BSA.
Add 20mM phosphate buffer (pH 6.0) containing 125I to make a total volume of 50μl, remove unbound 125I using a Sephadex G25 column, collect the void fraction, and remove 125I.
An I-activin-like substance liquid was obtained. The specific activity of 125 I-activin-like substance is 15 mCi/
It was mg. 0.5% BSA and 0.15M N
10mM phosphate buffer (pH 7.2) containing aCl (R
Example 10 in a test tube containing 0.5 ml of IA buffer)
0.1 ml of the monoclonal antibody obtained in (1) adjusted to 5 μl/ml with RIA buffer was added, and as a sample, the activin-like substance finally obtained in Example 2 was mixed with PBS- 0.1 ml of each diluted solution was added and incubated at room temperature for 16 hours. Then, 125 I-activin-like substance liquid (10
,000 cpm) and 0.1 ml of 100-fold diluted normal rabbit serum, further incubated overnight at room temperature, and 10-fold diluted goat anti-rabbit Ig.
100 μl of G serum was added. After overnight incubation at 4°C, radioactivity in the pellet was measured by centrifugation at 3000 rpm for 15 minutes. Using this measurement system, 5n
It was possible to detect activin-like substances of g/ml or more. (Example 16) Chemiluminescence immunoassay (1) Preparation of labeled antibody First, acridinium ester (chemical formula is shown in Chemical formula 1)
was dissolved in DMF to a concentration of 1 mM to prepare an acridinium ester solution. 10 μl of the above acrinidium ester solution and 75 μg of the monoclonal antibody obtained in Example 10 (1) were added to 0.01 M phosphate (pH
8.0) 500 μl of monoclonal antibody solution diluted with
and incubate for 15 minutes, then incubate for 10 m
200 μl of g/ml lysine solution was added and incubated for 15 minutes. After the incubation, the sample was pre-equilibrated with 0.1M phosphate (pH 6.3) using a Sephadex G50 column (manufactured by Pharmacia).
Served. Elution was performed with 0.1M phosphate (pH 6.3), a portion of each fraction was collected, and 0.1N nitric acid and 0.5% H2O2 were used as trigger solution 1, and 0.25N NaOH and 0.25N NaOH were used as trigger solution 2. 0.125%C
TAB was added and luminescent activity was measured. The fraction in which the strongest luminescent activity was observed was used as a labeled antibody in the following experiment. embedded image (2) Preparation of measurement system and measurement Example 8 (
The polyclonal antibody obtained in step 1) was diluted to 10 μg/ml with 0.1 M phosphate (pH 6.5), and 5 μg/ml was added to each well of a 96-well black plate (manufactured by Dynatec).
0 μl each was added and allowed to stand at room temperature for 2 hours to allow the polyclonal antibody to be adsorbed onto the plate surface. Next, the activin obtained in Example 3 was mixed with 0.01% Tween 20/0.
1%BSA/0.01%NaN3/0.1MNaCl
/0.1M phosphate (pH 6.3) to various concentrations, added 50 μl to each well, and incubated for 15 minutes. After incubation, 0.01% Twee
Washed 5 times with n20/PBS- followed by Example 16
50 μl each of the labeled antibody solution obtained in (1) was added and incubated for 15 minutes. After incubation, 0.
After washing 5 times with 01% Tween 20/PBS- and adding 100 μl each of the aforementioned trigger liquid 1 and trigger liquid 2, the amount of luminescence was measured using a chemiluminescence analyzer (LB952, manufactured by Bernardo). As a result, a positive correlation was observed between the amount of luminescence and the activin concentration when the activin concentration was 1 ng/ml or higher. (Example 17) Measurement by time-resolved fluorescence immunoassay (1) Preparation of BSA-mediated europium chelate-labeled antibody The monoclonal antibody obtained in Example 9 (1) was treated with pepsin and F. (ab')2 fragment was obtained. Using the obtained F(ab')2 fragment, a BSA-mediated europium chelate-labeled Fab' fragment was produced by the following method. First, 50mM sodium carbonate buffer (p
0.2 ml of 5 mg/ml BSA dissolved in H8.0)
100 μg/ml of Eu dissolved in ethanol (
III) 2,9 -[N,N',N,N'-Bis(3
,6-dioxaoctamethylene)bis
(aminomethyl)]-4,7-isoth
iocyano-1,10-phenanthroli
ne (europium (III) [2.2.phen]
Add 25 μl of cryptate chelate and incubate for 30 minutes at room temperature.
Incubated for 0 minutes. Next, add this reaction solution to 50 m
It was applied to a Sephadex G25 column (1 cmφ x 15 cm) equilibrated with M phosphate buffer (pH 7.0),
Gel filtration was performed to collect the protein fraction, and the collected fraction was further concentrated to 0.2 ml using Centricut (molecular weight cut off: 10,000, Kurabo Industries, Ltd.). Next, 50 μl of 0.1M phosphate buffer (pH 7.0) was added to this solution.
0.4 mg of sulfosucciimidyl-4-(
N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, Pierce Chemical Co.)
was added and reacted at room temperature for 1 hour. The sample after the reaction was transferred to a Sephadex G25 column (1 cmφ x 15cm) equilibrated with 0.1M phosphate buffer (pH 6.2).
m), gel filtration was performed, and the protein fraction was purified with europium (III) [2.2. phen ] Cryptate chelate-BSA-maleimide was recovered and concentrated to 0.1 ml using Centricut. Next, the above monoclonal antibody F(a
b') 1 mg of the 2 fragment was subjected to the method of Ishikawa et al. (Ishikawa et al., J.I.
mmunoassay, Vol. 4(3), p. p
．． 209-327, 1983) and F
An ab' fragment was prepared, and 0.1M phosphate buffer (pH 6.0) containing 1mM EDTA was added.
A 5 ml Fab' fragment solution was prepared. next,
To this solution was added europium (II) prepared as described above.
I) [2.2. [phen] Cryptate chelate-B
0.1 ml of SA-maleimide solution was added, and the mixture was stirred at 37° C. for 3 hours under a nitrogen atmosphere. After that, 0.1Mβ-
10 μl of mercaptoethanol was added and stirred at room temperature for 15 minutes, and further N-ethylmaleimide was added and stirred at room temperature for 15 minutes. 0.1M sodium carbonate buffer (pH 8.0) was added to the reaction mixture to make a total volume of 1.5 ml, and Ultragel ACA34 was equilibrated with the same buffer.
It was applied to a column (2.5 cmφ x 40 cm) and subjected to gel filtration to obtain a BSA-mediated europium chelate-labeled anti-activin monoclonal antibody Fab' fragment. BSA-mediated europium (III) [2.2phen] cryptate chelate-labeled anti-activin monoclonal antibody Fab thus prepared.
'The protein concentration of the fragment is 190 μg/ml, and the concentration as labeled europium is 2.1 x 10-5
It was M. (2) Preparation of measurement system and measurement Example 8 (
The polymonoclonal antibody obtained in 2) was added to 10 μg/ml in 0.1 M sodium carbonate buffer (pH 9.5).
This was prepared in a 96-well microtiter plate (E
Add 200 μl to each well of FLAB (manufactured by FLAB),
Incubated overnight at °C. The wells were then washed five times with deionized water and then washed with 0.5% BSA.
Blocking treatment was performed by adding 300 μl of 1M sodium carbonate buffer (pH 8.5) to each well. After washing the thus prepared anti-activin polyclonal antibody insolubilized well with deionized water, the activin finally obtained in Example 1 was added as a sample to the assay buffer (0.5% serum albumin, 0.0%
．． 50m containing 01% Tween20, 0.9% NaCl
200 μl of various concentrations prepared with M Tris-HCl buffer (pH 7.8) was added to the wells, and the mixture was allowed to react at room temperature for 1 hour with gentle shaking and stirring. Next, the wells were washed with a physiological saline solution containing 0.005% Tween20, and then the BSA-mediated europium (III) diluted with the assay buffer [2.2.
[phen] Cryptate chelate-labeled anti-activin monoclonal antibody Fab' fragment solution was added in an amount of 200 μl to each well, and incubated at room temperature for 1 hour. After washing each well, 200 μl of 50 mM Tris-HCl buffer (pH 8.0) containing 50 U/ml pronase was added to each well and incubated at 40° C. for 10 minutes to perform a desorption reaction. Then, 75 μl of 1M sodium acetate buffer (pH 3.5) was added to each well, and a time-resolved fluorometer (Arcus 1
Fluorescence intensity was measured using LKB-230 (manufactured by Wallac Co., Ltd.). According to this method, it was possible to measure activin at a concentration of 5 ng/ml or more. (Example 18) Measurement of urinary activin and/or activin-like substances by enzyme immunoassay Urine was collected from 5 male aplastic anemia patients with decreased platelet count and 5 healthy males, and after concentration. , the amount of activin and/or activin-like substance in the urine was measured by the solid phase sandwich method described in Example 12. Or a significant difference was observed in the amount of activin-like substances. (Example 19) Measurement of urinary activin and/or activin-like substances by radioimmunoassay Urine was collected from 7 male cancer patients with decreased platelet count and 7 healthy males, and after concentration, When the amount of activin and/or activin-like substance in the urine was measured by the radioimmunoassay described in 15, there was a significant difference in the amount of activin and/or activin-like substance in the urine between the cancer patient group and the healthy group. was recognized. (Example 20) Measurement of urinary activin and/or activin-like substances by chemiluminescence immunoassay Urine was collected from 5 idiopathic thrombocytopenia patients with decreased platelet count and 5 healthy males and concentrated. Later, when the amount of urinary activin and/or activin-like substances was measured using the chemiluminescence immunoassay described in Example 16, the amount of urinary activin and/or activin-like substance was determined between the idiopathic thrombocytopenia patient group and the healthy control group. Or a significant difference was observed in the amount of activin-like substances. (Example 21) Measurement of urinary activin and/or activin-like substances by time-resolved fluorescence immunoassay Urine was collected from 5 DIC patients and 5 healthy men.
After concentration, the amount of activin and/or activin-like substances in the urine was measured using the time-resolved fluorescence immunoassay method described in Example 17. Or a significant difference was observed in the amount of activin-like substances. (Example 22) Serum activin and/or
Or measurement by enzyme immunoassay of activin-like substances. Blood was collected from 5 male aplastic anemia patients with decreased platelet count and 5 healthy males, serum was prepared, and after concentration, the solid phase sandwich described in Example 12 was prepared. When the amount of activin and/or activin-like substances in the serum was measured using a method, a significant difference was found in the amount of activin and/or activin-like substances in the serum between the aplastic anemia patient group and the healthy group. It was done. (Example 23) Serum activin and/or
Alternatively, measurement of activin-like substances by radioimmunoassay. Blood was collected from 7 male cancer patients with decreased platelet count and 7 healthy males, serum was prepared, and after concentration, Example 1
When the amount of activin and/or activin-like substance in the serum was measured by the radioimmunoassay described in 5, there was a significant difference in the amount of activin and/or activin-like substance in the serum between the cancer patient group and the healthy group. was recognized. [0114] The present invention provides a method for measuring activin and/or an activin-like substance, which includes the step of using an antibody that recognizes at least a portion of activin and/or an activin-like substance. Since activin and/or activin-like substances are substances involved in the megakaryocyte-platelet system, by implementing the measurement method of the present invention or by providing a diagnostic agent for implementing the method, It becomes easier to predict and diagnose an increase or decrease in the megakaryocyte-platelet system or a functional abnormality in the megakaryocyte-platelet system, and to predict and diagnose diseases accompanied by these. In this manner, the present invention contributes to the prevention and treatment of increases or decreases in the megakaryocyte-platelet system, abnormalities in the function of the megakaryocyte-platelet system, and diseases accompanied by these. [Sequence Listing] SEQ ID NO: 1 Sequence Length: 20 Sequence Type: Amino Acid Topology: Unknown Sequence Type: Protein Origin Cell Type: THP-1 Sequence SEQ ID NO: 2 Sequence Length :24 Sequence type: Amino acid topology: Unknown Sequence type: Type of cell of protein origin: KHM-5M Sequence SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid sequence TGCTGCCAGG ATG CCC TTG C[
SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid sequence AACT CTA TGA GCA CCC ACA
C SEQ ID NO: 5 Sequence length: 10 Sequence type: Amino acids Topology: Unknown Sequence type: Protein source cell type: CHO cell Cell line: CHO DXB11 Sequence SEQ ID NO: 6 Sequence length S: 116 Sequence type: Amino acid topology: Unknown Sequence type: Protein sequence 0121 SEQ ID NO: 7 Sequence length: 115 Sequence type: Amino acid topology: Unknown Sequence type: Protein sequence

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the elution pattern of activity in anion exchange chromatography in Example 2 (2).

FIG. 2 is a diagram showing the DNA sequences of sense primer A and antisense primer A.

FIG. 3 shows the amino acid sequences of the βA chain and βB chain of activin AB.

Claims (14)

[Claims] 1. A method for measuring activin and/or an activin-like substance in a target sample, the method comprising the step of using an antibody that recognizes at least part of the activin and/or activin-like substance. 2. The measuring method according to claim 1, wherein the antibody recognizes part or all of at least one of activin A, activin B, and activin AB. 3. The measuring method according to claim 1, wherein the antibody recognizes part or all of activin A. 4. Used for predicting and diagnosing an increase or decrease in the megakaryocyte-platelet system and/or abnormal functioning of the megakaryocyte-platelet system, and for predicting and diagnosing diseases accompanied by these. 3. The measurement method according to any one of 3. 5. The measuring method according to claim 1, wherein the measuring method is an enzyme immunoassay. 6. The measuring method according to claim 1, wherein the measuring method is a radioimmunoassay. 7. The measuring method according to claim 1, wherein the measuring method is a chemiluminescence immunoassay. 8. The measuring method according to claim 1, wherein the measuring method is a fluoroimmunoassay. 9. The measuring method according to claim 1, wherein the measuring method is a time-resolved fluorescence immunoassay. 10. The measuring method according to claim 1, wherein the measuring method is an immunoassay using particle aggregation as an indicator. 11. The measuring method according to claim 10, wherein the particles are latex particles. 12. The measuring method according to claim 10, wherein the particles are red blood cell particles. [Claim 13]Claim 1, wherein the target sample is a body fluid.
13. The measuring method according to any one of 12 to 12. 14. The body fluid is blood, plasma, serum,
The measuring method according to claim 13, wherein the measuring method is selected from urine, cerebrospinal fluid, lymph fluid, saliva, ascites, and pleural fluid.