JPH04179496A - Monoclonal antibody specifically recognizing human carpine ii and hybridoma cell strain producing the same, and immunologically measuring method using the same - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody specifically recognizing human carpine II antibody produced with a hybridoma prepared by fusing a myeloma cell strain with a lymphocyte prepared from a mammalian immunized with a synthetic peptide containing an amino acid sequence specific to the human carpine as an antigen. USE:A reagent for the immunological measurement of human carpine II. A medicine for diagnosing and treating muscular diseases, etc. PREPARATION:For example, a substance formed by ligating human carpine II with bovine serum albumin (BSA) as a carrier protein is injected as an immunogen into the abdominal cavity of a BALB/c mouse. After the mouse is perfectly immunized, splenocytes are collected, fused with myeloma cells and subsequently cultured in an HAT medium. The obtained hydridomas are screened, and the separated carpine-specific antibody-producing clone is cloned by a limiting dilution method, etc., followed by culturing the clone to provide the objective monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a monoclonal antibody that specifically recognizes human calpain II, a hybridoma cell line that produces the same, and an immunoassay method using the same. More specifically, the present invention relates to a monoclonal antibody against human calpain II, a hybridoma cell line that specifically produces this monoclonal antibody, and a method for treating calpain in biological fluids using this monoclonal antibody useful for the diagnosis and treatment of muscle diseases. This relates to the immunoassay method described in (2).

(Prior art) Calpain, a Ca''+-dependent cysteine endopeptidase, is ubiquitous in various tissues, and exists in two types as inactive precursors in the cytoplasm of cells.These two types of enzymes, calpain I and calpain■ are Ca”+
These are isozymes with different requirements. The calpain precursor consists of two subunits with molecular weights of 80 and 30, and is activated when Ca''+ binds to the calmodulin-like Ca''+ binding region of each subunit.It becomes a substrate protein that is degraded by calpain. There are intracellular enzymes such as kinase, membrane proteins such as hormone receptors and Ca2+-ATPase, cytoskeletal and related proteins such as spectrin, membrane proteins, etc., and calpain is responsible for the decay phenomena of these proteins and protein functions. It causes an on-off phenomenon. Calpain is thought to be involved in the breakdown of skeletal muscle seen in muscle diseases such as muscular dystrophy through the decomposition of muscle fiber proteins. (Akira Ishiura et al., Metabolism Special Issue [Metabolic Disease Highlight'p] 25.1988).

Calpain I and calpain ■ are widely distributed in various tissues in the body, and the abundance of these isozymes differs depending on the organ or tissue, and the state of distribution changes uniquely depending on the pathological condition and the like.

Therefore, by separately quantifying calpain ■ and calpain ■ in each tissue or biological fluid and knowing the distribution state of each, it becomes possible to understand the physiological function of calpain and furthermore diagnose muscle diseases.

Traditionally, to quantify calpain in biological fluids,
Enzyme-chemical methods are used. For example, a specimen containing calpain is reacted with casein as a substrate under certain conditions, and trichloroacetic acid (T
After stopping the reaction with CA), the amount of peptide in the reaction filtrate is measured to determine the activity of calpain, thereby quantifying the calpain content in the sample.

(Problem to be Solved by the Invention) However, in such conventional quantitative methods, it is impossible to distinguish whether the measured activity value is due to the enzymatic activity of calpain I or calpain II, which are isozymes. In order to clarify this, it was necessary to separate calpain I and calpain ■ from a large amount of specimens by chromatography. Furthermore, under such measurement conditions, unlike in biological fluids, there is a problem that calpain's Ca2+ requirement, autolysis, etc. affect enzyme activity.

In this way, in the case of the conventional method for measuring calpain■,
Since the content of calpain II in the sample is determined by enzyme activity under conditions different from those in the living body, there was a drawback that the measured value was inaccurate.

Generally, when quantifying a specific physiologically active substance in a living body,
If an antibody, particularly a monoclonal antibody, for the substance is identified, it becomes possible to directly quantify the target substance in an immunological assay by adding the antibody bound to a label to a sample, for example. However, until now, no specific monoclonal antibody against human calpain II has existed.

This invention has been made in view of the above circumstances, and aims to provide a monoclonal antibody against human calpain (2), which has not existed up to now. Another object of the present invention is to provide a hybridoma cell line that specifically produces this monoclonal antibody and an immunoassay method for human calpain II using this monoclonal antibody.

(Means for Solving the Problems) The present invention solves the above problems by providing lymphocytes prepared from a mammal immunized with a peptide synthesized with an amino acid sequence site specific to human calpain II as an antigen; Human calpain I, characterized in that it is produced from a hybridoma fused with a myeloma cell line.
A monoclonal antibody that specifically recognizes I is provided. This invention also provides that the amino acid sequence of the peptide synthesized with the amino acid sequence site specific to human calpain II is Tyr-As.
p-I 1e-8er-Glu-Asp-Asp-I
A preferred embodiment is 1e-Asp-ASI)-Gly-Val-Cys.

Furthermore, this invention provides a hybridoma cell line capable of producing the monoclonal antibody that specifically recognizes human calpain
provides an immunoassay method for

This invention will be explained in detail below.

In this invention, a highly hydrophilic amino acid sequence site specific to calpain II is selected from the amino acid sequence of human calpain ■, for example, Tyr-Asp-Ile-
8er-Glu-Asp-Asp-I 1e-Asp-
A peptide having an amino acid sequence consisting of Asp-Gly-Vat-Cys is synthesized and used as an antigen, and a monoclonal antibody against it is prepared. Therefore, there is no need to directly collect calpain from human biological fluids or tissues, solving the problem of cross-reactivity and making it possible to quantify calpain directly.

Therefore, the monoclonal antibody of the present invention can be obtained by selecting and culturing a hybridoma of myeloma cells and lymphocytes prepared from a mammal immunized with a synthetic peptide antigen having the amino acid sequence as described above. From the point of view of production efficiency, among the above hybridomas, cells that specifically produce monoclonal antibodies against calpain II are selected, cloned to form a cell line, and cultured in this cell line to produce human calpain II. How to obtain monoclonal antibodies? Preferable methods for creating such cell lines and collecting monoclonal antibodies, and human calpain using the monoclonal antibodies.
The immunoassay method for can be performed as follows.

(1) Creation of a hybridoma cell line that specifically produces a monoclonal antibody against human calpain ■: First, in order to create a hybridoma, lymphocytes of a mammal immunized with a synthetic peptide antigen having an amino acid sequence specific to human calpain II, Prepare myeloma (myeloma cells) to fuse these.

In order to collect the above-mentioned lymphocytes, first, a calpain antigen consisting of a complex (hereinafter referred to as a calpain low carrier) in which a synthetic peptide with a human calpain II-specific amino acid sequence site and a carrier protein are covalently bonded is prepared. and immunize a mammal, preferably a mouse or rat, with this.

Immunization methods such as the amount of antigen to be used, the site of administration, and the use of adjuvants may be in accordance with conventional methods for obtaining antiserum. For example, when using mice, 0.001 to long, preferably 0.01 to 1 mg of calpain low carrier per mouse is mixed thoroughly with an adjuvant (for example, Freund's complete adjuvant) at the first time.
Administer subcutaneously, intraperitoneally, etc., and after 2 weeks or more, add an adjuvant (e.g., Freund's incomplete adjuvant) again.
Mix well and administer subcutaneously, intraperitoneally, etc. moreover,
After 2 weeks or more, calpain low carrier alone is administered intravenously, subcutaneously, intraperitoneally, etc. to sufficiently immunize the animal. Animals thus immunized are preferably immunized two days after the final immunization.
After ~4 days, sacrifice and collect lymphocytes. Spleen, lymph nodes, peripheral blood, etc. are used for lymphocyte preparation. The lymphocytes are suspended in a culture medium.

On the other hand, it is preferable to use myeloma derived from the same species as the immunized animal. Furthermore, the myeloma is preferably a drug-resistant mutant strain, and preferably is an unfused myeloma or one that does not grow in a hybridoma selection medium. Most commonly, 8-azaguanine resistant cell lines are used. This is hypoxanthine-quanine-phosphoribosyltransferase (1).
nt inguanine phosphorjbos
yl transferase) and cannot grow on hypoxanthine-aminopterin-thymidine (HAT) medium, a type of selective medium. In addition, it is preferable that the myeloma itself is one that does not secrete antibodies. From the above points, for example, commercially available mouse myeloma P3, X63,
Ag8.6.5.3 (X63.6.5.3); P3.X
It is preferable to use 63.Ag8.Ul (P3U1), rat myeloma 210-RCY3.Agl.2.3, and the like.

The mini-Roma was placed in Eagle's minimal medium (MEM) containing serum, preferably fetal bovine serum, RP MI 16.
40 medium (RP MI 1640) or the like.

Next, the lymphocytes and myeloma obtained above were each suspended in a medium such as MEM, RPM 11640, etc.
Mix. At this time, can the mixing ratio of - be selected arbitrarily?
Preferably lymphocytes: Myeloma is 1°1 to 2 cells in number.
A ratio of 0:L, preferably 5:1 to 1O:1 may be used. The mixed cells are fused using a fusion promoter.

As a fusion method, for example, Immunologica
l Methods Val, U, 285. 1
981° Academic Press. As the fusion promoter, various polymeric substances, viruses, etc. can be used, but polyethylene glycol (PEG) and Sendai virus are preferably used. PEG having an average molecular weight of 400 to 20,000 can be used, but preferably 1,000 to 7.50.
0 may be used. The concentration used is 40-60
Vol1. % is preferred.

The fused cells are washed to remove the fusion promoter, and then washed for 5 to 1
5Vo1. MEM or RP MI containing % serum
Suspend in 1640 medium and 0.5-5X in a 96-well culture dish etc.
106. Dispense in proportion to the hole. Furthermore, if a selective medium (for example, HAT medium) is added to each person and the selective medium is replaced as appropriate, unfused myeloma will die after 10 to 14 days, and only hybrid l''-ma will grow. Lymphocytes cannot be grown in vitro for long periods of time and die after 10-14 days.

Next, hybridomas that produce antibodies against calpain H are searched and selected. For that purpose, EL
The ISA method can be used. However, in that case, since the above hybridoma uses calpain low carrier as an antigen, in addition to producing antibodies against calpain ■ synthetic peptide, there are also hybridomas that produce antibodies against carrier protein, so they will produce antibodies against calpain II. Only hybridomas need to be selected.

To do this, for example, a complex (hereinafter referred to as calpain low carrier 2) in which calpain ■ synthetic peptide is covalently bonded to a protein other than the carrier protein is created, this is adsorbed onto an ELISA plate, and a hybridoma is attached to this. After adding the supernatant and washing, add a commercially available labeled antibody to immunized animal immunoglobulin (for example, horseradish peroxidase (HRP) labeled antibody or +2
J-labeled antibody). As a result, if an antibody against calpain synthetic peptide was present in the hybridoma supernatant, it was detected that it was absorbed by the solid-phase calpain raw carrier 2.
A labeled antibody against the immunoglobulin binds to this, and a signal is generated by the label. on the other hand,
If there is no antibody against calpain II in the supernatant, nothing will bind to the solid-phase calpain low carrier 2, and therefore no signal will be obtained. In this way, hybridomas can be selected using the presence or absence of a signal from a label as a clue.

Next, a hybridoma cell line can be created by selectively culturing the thus obtained hybridoma that specifically produces the monoclonal antibody against calpain H.

In addition, according to this method, one type of hybridoma cell line was created by fusing mouse lung lymphocytes and mouse myeloma cells that had been immunized with a calpain low carrier in advance.
The hybridoma was named CALn3GO5. On August 27, 1990, procedures were carried out to deposit this strain with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
The acceptance was made as No. 98 (FERM P-11698). This CALn3GO5 can be cryopreserved almost permanently at -120°C or lower, and is always available for distribution.

This hybridoma CAL113GO5 can be grown in a commonly used medium. For example, fetal bovine serum
Using RPM I 1640 or MEM containing 20% as a medium, the temperature was 37°C and the carbon dioxide concentration was 5 Vol. It grows well in air containing %. It also has myeloma tumorigenic properties, so it can proliferate in vivo (eg, in syngeneic animals, nude mice, etc.) and produce monoclonal antibodies against calpain H.

(2) Collection of large amounts of monoclonal antibodies against calpain II: By culturing the hybridoma cell line prepared as described above, monoclonal antibodies against calpain II can be collected in large quantities.

There are two main methods for collecting monoclonal antibodies. One method is to culture in a culture container such as a flask using a medium, and collect the antibody from the supernatant. For example, 5 to 10 Vo1. M containing % serum
0.5-5X10' in EM or RPM11640 medium
10 hybridoma cell lines can be planted in 2-4 days.
This is a method in which antibodies are collected from the supernatant after the culture is grown to ~20 times. Another method is to inoculate the hybridoma cell line thus cultured in a culture vessel into a syngeneic animal. That is, a method in which 10 to 10 hybridoma cell lines are administered subcutaneously or intraperitoneally to a syngeneic animal, and after 7 to 20 days, when the hybridoma cell lines proliferate and the tumor becomes large, serum and ascites are collected. It is. When administering intraperitoneally, pre-administer (3 to 3 minutes)
7 days ago) 2. 6. 10. When mineral oils such as 14-tetramethylpentadecane are administered, larger amounts of ascites are obtained.

The antibody thus obtained can be purified and used if necessary. For example, purification can be carried out using means commonly applicable to proteins, such as ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography with immobilized protein A.

In this way, monoclonal antibodies against calpain II can be easily obtained in large quantities.

(3) Immunological assay of calpain ■ using monoclonal antibody: First, calpain ■-carrier is immobilized on a solid phase. As the solid phase, plastic test tubes, microtiter plates, glass beads, plastic beads, membranes, etc. can be used.

A general physical adsorption method can be used to immobilize the calpain -carrier, but a covalent bonding method can also be used when immobilizing it on a solid phase having a functional group. At this time, the concentration of calpain ■-carrier is 0.0
01 .mu./- or more, preferably 0.05 to 0.2 .mu./-, and may be brought into contact with the solid phase.

Next, several kinds of liquids with known concentrations of calpain (2) or samples with unknown concentrations of calpain (2) are added to the solid phase on which the calpain (2) carrier is immobilized in this manner.

Further, an antibody against calpain II labeled with an enzyme, RI, a fluorescent substance, etc. is added, or after adding an antibody against calpain II, a secondary antibody labeled with an enzyme RI, a fluorescent substance, etc. is added.

Note that, as the enzyme, HRP, bovine small intestine alkaline phosphatase, etc. can be used. Also, as an RI +
251, fluorescein isothiocyanate, tetramethylrhodamine isocyanate, etc. can be used as a fluorescent substance.

Next, a calibration curve is created by measuring the signals of several types of labeled antibodies in liquids with known concentrations of calpain, and the signal obtained from a sample with unknown calpain concentration is applied to this calibration curve to determine the calpain concentration of the sample. Quantify.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 (Immunogen and hybridoma cell line screening) As a carrier protein, Keyhole Lympet
Homocyanin (K L H) and bovine serum albumin (BSA) were used to screen immunogens and hybridoma cell lines.

Dissolve 10■KLH or 20■BSA in 2.51nl of O.1M sodium phosphate buffer (pH 7.0),
Add 250 μl N, N-dimethylfor
2.5 mg N-Hydrox dissolved in mamide
ysuccimido ester ofN -(4-
carboxy cyclohexyl methyl)
Maleimide was added and reacted at a temperature of 30° C. for 30 minutes to introduce a maleimide group into KLH or BSA. Next, these maleimide-KLH or maleimide-BSAm solutions and calpain ■ synthetic peptide 4 m
g dissolved in 0.1M sodium phosphate buffer (pH 6,0) containing ImMEDTA, and mixed.
Calpain ■-KLH or calpain I[-BSA was prepared by reacting at °C.

Furthermore, the maleimide group introduced into KLH or BSA will be measured by Eiji Ishikawa, Enzyme Immunoassay Method Igaku Shoin, p75.
), it is thought that all of these maleimide groups and the thiol groups of the synthetic peptide are bonded, so there are about 20 molecules of calpain synthetic peptide bound per molecule of KHL, and about 20 molecules of calpain synthetic peptide bound per molecule of B5Al. Approximately 7 molecules were determined.

Example 2 (Creation of hybridoma) Calpain ■ synthetic peptide-KLH prepared in Example 1 was added to 8-week-old mouse Ba1b/c (obtained from Oriental Bioservices) with complete Freund's adjuvant (obtained from Hani Chemical) and 1: 1 and administered intraperitoneally. Two weeks later, 501, zg of calpain synthetic peptide - KLH was intravenously injected for boosting. Three days later, the lungs were removed and placed in MEM medium (obtained from Hanui Chemical). It was loosened and washed in suspension. On the other hand, mouse myeloma X63.6.5.
3 (obtained from Kyoto University) was cultured for 2 days, and cells in the logarithmic growth phase were collected by centrifugation.

10' lung cells as myeloma X63, 6, 5, 3XIO
50% PEG 4000-RPM I 1 in a 37°C water bath after mixing with
640 (obtained from Kibuco) was gradually added over 1 minute, and after stirring gently for another 1 minute, 9-RPM
I 1640 medium was gradually added to dilute the PEG4000. The PEG solution was removed by centrifugation, and 201 nl of HAT medium containing 10% tallow serum was added to the pellet, which was then dispensed into two 96-well culture dishes (manufactured by Corning) in 0.1-ml portions for each person. Half of the culture medium was discarded three times on days 4, 8, and 11, and fresh HAT medium was added.

As a result, hybridoma growth was observed in 86 out of 384 wells after 10 days.

Example 3 (Creation of hybridoma cell line) Among the hybridomas obtained in Example 2, strains producing antibodies against calpain ■ synthetic peptide were searched for using ELISA.

First, 50 m on an ELISA plate (manufactured by Corning)
50 μβ of calpain synthetic peptide-BSA dissolved in M sodium carbonate buffer, pl (9,6) was dispensed to each person and left at a temperature of 25°C for 2 hours. After removing this solution, each person 20mM sodium phosphate buffer (pH 7) containing 1.0% BSA, 0.15M sodium chloride
, 2) and left at a temperature of 25° C. for 1 hour. Furthermore, this liquid was removed and 0.2% Tween 20.0.2
Wash each well well with a washing solution consisting of 20mM sodium phosphate buffer (pH 7.2) containing % BSA and 0.15M sodium chloride, and wash the calpain synthetic peptide-B.
An SA immobilization plate was created.

Next, 50 μl of the hybridoma supernatant obtained in Example 2 was added to this plate, and after leaving it at a temperature of 25°C for 2 hours, it was thoroughly washed with a washing solution, and HRP-labeled goat anti-mouse IgG (purchased by Funakoshi Yosu) was added. lμg/- to each person 50μ
1 was added and left at a temperature of 25°C for 2 hours. Furthermore, after washing, peroxidase measurement reagent (manufactured by Biorat) 50
Add μl and incubate for 20 minutes at a temperature of 25°C.
Peroxidase activity was stopped by the addition of 50 μl of %5DS and the absorbance at 415 nm (A 415)
was measured. In addition, as a control, a reaction using only HAT medium instead of the hybridoma supernatant was used.

As a result, a superior groove was obtained that showed a higher signal than the signal when no supernatant was added, which was used as a control. For these supernatants that showed high signals, human calpain II-immobilized plates were similarly prepared and the absorbance was measured by the same procedure, and supernatants that also showed high signals were obtained. A strain in the well of this supernatant was selected and cloned by limiting dilution method to obtain monoclonal hybridoma cell line CALn3G05.

Example 4 (Collection of monoclonal antibody against calpain II) The hybridoma cell line CALII3GO5 obtained in Example 3 was cultured, and a monoclonal antibody against calpain II was collected.

First, CALII3GO5 was added to RP containing 10% beef tallow serum.
Cultured in M I 1640 medium to a cell concentration of 2 x 10
The 300-culture that reached 6 cells/- was centrifuged, and the supernatant was subjected to 50% saturated ammonium sulfate fractionation to separate the crude antibody fraction.
After dialysis, Protein A-Sepharose (pH 9,0
) and eluted with citrate buffer (pH 3,0) to prepare an antibody against calpain II (hereinafter referred to as CALnA3G).
05) was obtained.

Next, human calpain II of this CALIIA3GO5
We investigated the specificity of

First, human calpain I and human calpain II solutions (0
,0280=0.05) on the ELISA plate.
2.0 μg/'-concentration of CALIIA3G was injected into each individual on the fixed ELISA plate.
0550μ! was added. After incubating the plate for 2 hours at a temperature of 25°C, 0.2% Tween 2
After thorough washing with 20 sodium phosphate buffer containing 0.0.2% BSA and 0.15 sodium chloride, HRP-labeled goat anti-mouse IgG antibody was added to each individual. Next, this was washed, 50 μl of peroxitase measurement reagent (manufactured by Biorat) was added, and the mixture was allowed to react at a temperature of 25°C for 20 minutes. After that, 50 μl of 10% 5DS was added to stop the reaction, and the absorbance at 415 nm was measured. .

As a result, monoclonal antibody CALIIA3G05 recognized human calpain ■,
It was also found that although human calpain ■ could be recognized slightly, the recognition ability was about 1/100 of that for human calpain H.

From the above, the monoclonal antibody CAL of this invention
IIA3GO5 is believed to be a specific monoclonal antibody against human calpain II.

(Effects of the Invention) As explained in detail above, this invention makes it possible to immunologically measure human calpain ■ using a monoclonal antibody, and to easily and quickly measure calpain ■ in biological fluids.
Moreover, it can be quantified with high precision.

Moreover, by establishing a hybridoma cell line that specifically produces a monoclonal antibody against human calpain II, it becomes possible to easily obtain a large amount of a monoclonal antibody against human calpain II, which is useful for diagnosis and measurement.

Claims (4)

[Claims] (1) A human body characterized by being produced from a hybridoma obtained by fusion of a myeloma cell line and lymphocytes prepared from a mammal immunized with a peptide synthesized with an amino acid sequence site specific to human calpain II as an antigen. Calpain II-specific recognition monoclonal antibody. (2) The amino acid sequence of the peptide synthesized with the amino acid sequence site specific to human calpain II is Tyr-Asp-
He-Ser-Glu-Asp-Asp-Ile-A
sp-Asp-Gly-Val-Cys (
1) Human calpain II-specific recognition monoclonal antibody described above. (3) Human calpain according to claims (1) and (2)
A hybridoma cell line capable of producing II-specific monoclonal antibodies. (4) Human calpain according to claims (1) and (2)
Human calpain using II-specific recognition monoclonal antibody
II immunoassay.