JPH0630620B2 - Monoclonal antibody reactive with cholinesterase having a molecular weight of 70,000 to 300,000 present in human body fluid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to cholinesterase (Small Cholinesterase; hereinafter abbreviated as “SCE”) having a molecular weight of 70,000 to 300,000 present in human body fluid.
To a monoclonal antibody that reacts with.

[Prior Art] Cholinesterase (3.1.1.8) is an enzyme that hydrolyzes choline ester into choline and organic acid. Serum cholinesterase is not only acetylcholine,
A non-specific enzyme (Pseudo-cholinesterase) that also thaws various choline esters and non-choline esters,
It is a glycoprotein containing sialic acid and having a molecular weight of 440,000 (tetramer), 220,000 (dimer) and 110,000 (monomer). As a measuring method, acetylcholine, acetylthiocholine, butylcholine, butylthiocholine and the like are used as substrates, and acetic acid or choline produced by the action of serum and thawing is measured.

[Problems to be Solved by the Invention] Since serum cholinesterase is produced in the liver and supplied to serum, its activity is sharply decreased in the case of liver parenchymal injury, and its measurement is performed as one of important liver function tests. Has been. Since cholinesterase is present in arteriosclerotic lesions where cholesterol ester is accumulated, it may be possible that cholinesterase may act on accumulated lipids, which may contribute to lipid metabolism and further to arteriosclerosis. It has been suggested and the research is drawing attention.

However, the physiological role of cholinesterase has not been fully clarified due to the absence of short-chain fatty acid esters, which are substrates for cholinesterase, in blood [Kutty, KM, Review, Biological funition of c
holinesterase.Clin.Biochem.13,239-243 (1980)]. Furthermore, the physiological role of the monomeric and dimeric cholinesterases, which have 10% or less of the total activity, is completely unknown.

Therefore, it is required to establish a separation and purification technique and a measurement technique for further detailed investigation of the properties of cholinesterase and analysis of the action mechanism.

The present invention has been made from the above viewpoints, and an object of the present invention is to provide a monoclonal antibody that reacts with SCE present in human body fluids.

[Means and Actions for Solving the Problems] In order to achieve the above object, the present invention is produced by a fused cell of an immune cell of a mammal immunized with cholinesterase and a myeloma cell of a mammal, and is produced in a human body fluid. Existing S
The subject is a monoclonal antibody characterized by reacting with CE.

Then, by using the monoclonal antibody obtained by the present invention, it was possible to purify and measure SCE immunologically.

The monoclonal antibody of the present invention is produced by cell fusion of mammalian immune cells immunized with cholinesterase and mammalian myeloma cells, and is most characterized in that it has specific reactivity with SCE.

Hereinafter, the monoclonal antibody of the present invention will be further described in detail.

Cholinesterase used as an immunogen is prepared by the method described in the literature [Bioche
mica et Biophysica Acta, 570 (1979) 88-95 etc.] and SC
Any product containing SCE, such as a purified product of E, can be used. The mammal immunized with the immunizing antigen is not particularly limited, but it is preferable to select it in consideration of compatibility with plasma cell type cells used for cell fusion, and in general, mouse, rat Etc. are advantageously used.

Immunization is carried out by a general method, for example, by administering the above-mentioned immune antigen to a mammal by intravenous, intradermal, subcutaneous or intraperitoneal injection. More specifically, the immunogen is administered to the animal several times every 2 to 14 days, optionally in combination with a conventional adjuvant to give a total dose of about 100 to 1
It is preferable that the amount is about 000 μg / mouse. As the immune cells, it is preferable to use spleen cells extracted about 3 days after the final administration.

In addition, as a mammalian plasma cell type cell as the other parent cell to be fused with the above-mentioned immune cell, various known cell lines such as p3 (p3 / x63-Ag8) [Natur
e, 256,495-497 (1975)], p3-U1 [Current Topics i
n Microbiology and Immunology, 81, 1-7 (1978)], NS
-1 [Eur.J.Immunol., 6,511-519 (1976)], MPC-1
1 [Cell, 8,405-415 (1976)], SP-2 / O [Nature, 2
76,269-270 (1978)], FO [J.Immunol.MeTH., 35,1-21
(1980)], x63.6.5.3. [J. Immunol., 123, 154
8-1550 (1979)], S194 [J.Exp.Med., 148,313-323 (1
978)], etc., and R210 in rats [Nature, 277,131
-133 (1979)] and the like are used.

The fusion reaction between the immune cell and the plasma cell type cell is basically carried out according to a known method, for example, the method of Milstein et al. [Method in Enzymology, Vol.73, pp3 (1981)] and the like. I can do it. More specifically, the fusion reaction is
For example, in a normal nutrient medium in the presence of a fusion promoter. As the fusion promoter, a commonly used one, for example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used, and if desired, an auxiliary agent such as dimethyl sulfoxide may be added to increase the fusion efficiency. it can. The use ratio of immune cells to plasma cell type cells is the same as in the usual method, and for example, immune cells may be used in an amount of about 1 to 10 times that of plasma cell type cells. The medium at the time of the fusion includes, for example, RPMI-1640 medium used for the growth of the plasmacytoplasmic cell line,
MEM medium and various other ordinary mediums used for cell culture of this type can be used, and it is usually preferable to remove serum supplement such as fetal calf serum (FCS). The fusion is performed by thoroughly mixing a predetermined amount of the above-mentioned immune cells and plasma cell type cells in the above medium and preliminarily heating to about 37 ° C. in a PEG solution, for example, one having an average molecular weight of about 1,000 to 6,000, It is usually carried out by adding to the medium at a concentration of about 30 to 60 W / V% and mixing. After that, a desired hybridoma is formed by repeating the operation of sequentially adding an appropriate medium, centrifuging and removing the supernatant.

Isolation of the desired hybridoma obtained is carried out by culturing in a usual selection medium, for example, HAT medium (medium containing hypoxanthine, aminopuritene and thymidine). Culturing in this HAT medium may be carried out for a time sufficient to kill cells other than the target hybridoma (unfused cells etc.), usually for several days to several weeks. The hybridoma thus obtained is subjected to the usual limiting dilution method,
Search and monocloning of the target antibody producing strain are performed.

The production strain can be searched by, for example, the ELISA method [Engvall,
E., METH.Enzymol., 70,419-439 (1980)], plaque method,
Spot method, agglutination method, Ouchterlon
y) method, radioimmunoassay (RIA) method, and various other methods generally used for antibody detection [[Hybridoma method and monoclonal antibody], R & D Planning, Inc., pp30-53, March 5, 1982. ] It is performed according to. Also, this search is performed using the immunogen.

The thus-obtained hybridoma producing an antibody confirming SCE can be subcultured in an ordinary medium,
It can be stored in liquid nitrogen for a long time.

The monoclonal antibody of the present invention can be collected from the hybridoma by culturing the hybridoma according to a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal having compatibility with the hybridoma and proliferating the hybridoma as ascites. The method of obtaining it is adopted. The former method is suitable for obtaining highly pure antibody, and the latter method is suitable for mass production of antibody. The antibody obtained as described above can be further purified by a conventional purification means such as salting out, gel filtration, affinity chromatography and the like.

Since the monoclonal antibody of the present invention has extremely high specific reactivity with SCE, it can be used for purification and measurement of SCE. Therefore, the present invention provides an immunological purification method of SCE and SC using the above-mentioned specific antibody.
It also provides an immunological assay for E.

Purification and measurement of SCE are carried out by a conventional method except for the use of the monoclonal antibody of the present invention. Hereinafter, the measuring method of SCE will be described in detail.

SCE can be measured by known conventional immunoassay methods such as RI.
Method A, enzyme immunoassay (EIA), etc., and the operations, procedures, and the like in each of these immunoassays are not particularly different from those generally adopted, and include, for example, known direct methods, indirect methods, and competitive methods. Method, sandwich method and the like.

For the purpose of diagnosis, cells, tissue pieces and / or various body fluids are used as specimens in the above-mentioned assay method.
When cells and / or tissue pieces are used as a specimen, the method is carried out according to the usual direct or indirect immunization method. According to this method, physiological saline or normal phosphate buffer (PB
S) or the like, or the cells or tissue slices fixed on a glass slide or the like are immunoreacted with the cells liberated in the buffer, and the cells or tissue pieces are thoroughly washed with the above buffer. The presence of the monoclonal antibody of the present invention bound to cells or tissue fragments may be assayed directly or indirectly as usual. As the labeling agent for the above-mentioned assay, a part of which will be described later, ordinary fluorescence or enzyme can be used.

When a body fluid is used as the measuring material, the usual method can be followed. Here, as the body fluid, for example, blood, cell tissue fluid, lymph fluid, pleural fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, spinal fluid, saliva, etc., or centrifugal supernatant after solubilization of the cells or tissue pieces is used can do.

Insolubilization method (method using insolubilized antigen or insolubilized antibody)
In the case of employing, the SCE or the monoclonal antibody of the present invention is produced by reacting an insoluble carrier chemically or physically according to a conventional method. Examples of the non-solvent carrier include cellulose powder, sephadex, sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid. A copolymer, an amino acid copolymer, an ethylene-maleic acid copolymer, etc. can be used. For insolubilization, the diazo method as a covalent bond method, the peptide method (acid amide derivative method, carboxy chloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbonate method) Derivative method, method using condensation reagent, etc.), alkylation method, carrier binding method using cross-linking reagent (glutaraldehyde, hexamethylene isocyanate, etc. are used as cross-linking reagent), Ug
It is carried out by a chemical reaction such as a carrier binding method by i reaction, an ionic binding method using a carrier such as an ion exchange resin, or a physical adsorption method using porous glass such as glass beads as a carrier.

As the labeled antigen or labeled antibody, the SCE or the monoclonal antibody of the present invention labeled with various labeling agents such as usual radioactive substances, enzyme labeling substances and fluorescent substances is used. As the radioactive substance as the labeling agent, 125
Radioactive iodine such as -I is used as a fluorescent substance, and fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRIT) is used.
C), substituted rhodamine isothiocyanate (XRIT
C), rhodamine B / isothiocyanate, dichlorotriazine fluorescein (DTAF) and the like, and as enzyme labeling substances, peroxidase (POX), alcalphosphatase, β-galactosidase, microperoxidase, chymotrypsinogen, procarboxypeptidase. , Glyceroaldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-nase,
R-nase and the like can be mentioned respectively. The labeling method by these can also follow a conventional method [J. Biol, Chem., 254, 9349-9351 (1979); Nature, 194, 495.
(1962); fluorescent antibody method, medical chemistry experiment course NO.4,263-270; Ac
ta, Endocrinol.Suppl.168,206 (1972); Proc.Nat.Acad.S
ci., USA, 57, 713 (1967), etc.].

The method for measuring and quantifying the test substance SCE in the sample in the present invention is carried out by immunoreacting with any of the above-mentioned insolubilized antigen, insolubilized antibody, labeled antigen and labeled antibody. In this case, the solvent used in the measurement system is an ordinary solvent that does not adversely affect the reaction, such as citrate buffer, phosphate buffer, Tris-hydrochloric acid buffer, borate buffer, acetate buffer, etc. A buffer solution of about 8 can be exemplified as a preferable one. In addition, the immune reaction conditions at the time of measurement are not particularly limited, and may be the same as the usual measuring method of this type. That is, the immune reaction is generally carried out at a temperature of 45 ° C or lower, preferably about 4 to 40 ° C, for 1 to 40 hours. Separation of the conjugate and the free form (BF) after completion of the immune reaction also follows a known method, for example, when the insolubilization method is adopted, solid phase-by separation means such as centrifugation, filtration, washing and decantation. The liquid phase can be separated. In other cases, for example dextran-
Conventional methods such as the activated carbon method and the second antibody method may be used.

A particularly convenient way to carry out the above assay is by using a kit. It is important that such a kit contains the monoclonal antibody of the present invention as an antibody reagent. Stabilizers and / or preservatives such as glycerol and bovine serum proteins can be added to this antibody reagent. Preferably, this antibody reagent is freeze-dried, and the kit may contain a water-soluble or water-miscible solvent. Furthermore, this antibody reagent
Buffers to keep the reconstituted reagent system at a constant pH and / or preservatives and / or stabilizers to prevent deterioration of the sample prior to use can be added. Although the buffer solution is not considered to be an essential component of the kit reagent, it is preferable to use a buffer solution having a pH of about 4 to 8 when carrying out the above measurement method. Further, the reconstitution agent preferably contains water, but a part or all of water can be replaced with a solvent miscible with water. Solvents miscible with water are well known to those skilled in the art, and for example, glycerin, alcohols, glycols, glycol ethers and the like can be used.

[Example] 1; Purification of immunogen 200 m of fresh human serum was added to 10 mM potassium phosphate buffer
It was dialyzed against (pH 8.0) at 4 ° C. or lower for 24 hours. next,
Trimethy1 ammonium anili was equilibrated with the same buffer.
It was applied to a Sepharose column (2.5 × 10 cm) bound with 230 mg of nium, washed with 200 m of the same buffer, and then the target enzyme was eluted with the same buffer containing 200 mM NaCL. 50 mM NaCL
After dialysis with 10 mM phosphate buffer containing Trimethy
It was applied to 1 ammonium anilinium Sepharose column, washed with 200 m of the same buffer, and then a concentration gradient from 0 mM to 200 mM NaCL was applied to separate the fraction containing the target enzyme. Furthermore, this was dialyzed with a 10 mM potassium phosphate buffer solution, equilibrated with the same buffer solution, and then subjected to DEAE-Sephadex column (1.2 × 16 c).
m), 200m from 10mM potassium phosphate buffer
A concentration gradient was applied to the M phosphate buffer solution to obtain 0.5 mg of cholinesterase containing SCE. This was stored at 20 ° C. or lower after dialysis and concentration.

2; Preparation of hybridoma 1: 1 immunogen and Freund's complete adjuvant
250 μl (immunogen 20 μg) to Balb / c
An intradermal injection was given to the back of a mouse (♀, 8 weeks old). Furthermore, 2
The same amount was immunized 6 times at weekly intervals. Three days after the final immunization, the spleen is removed and the splenocytes are washed 3 times with RPMI-1640 medium. After similarly washing the mouse myeloma cell line NS-1, the NS-1, 2.2 × 10E7 cells and the spleen cells (1.6 × 10E8) are placed in a 50 m centrifuge tube and mixed.
After centrifugation at 200 xg for 5 minutes, the supernatant is removed with a Pasteur pipette. Polyethylene glycol 4 kept at 37 ℃
000 (manufactured by Sigma) 50 w / v% RPMI-164
0.5m of 0 solution is added dropwise over 1 minute and mixed slowly for 7 minutes. RPMI-1640 (15% FCS, 1 mM pyruvate kept at 37 ° C. (hereinafter “complete RPMI-1
640 ") is added for 1 minute, the same amount of complete RPMI-1640 is added for 1 minute, then 8 m of complete RPMI is added dropwise, and the mixture is slowly stirred for 2 minutes. Two
After centrifuging at 00 × g for 5 minutes, the supernatant was removed, and the cells were suspended in complete RPMI-1640 kept at 37 ° C. at a cell concentration of 1 × 10E7 cells / m, and 100 μm on Microtest-I plate (Falcon). Each is inoculated and cultured at 37 ° C. in a 5% carbon dioxide gas incubator. 24 hours later 1.0x
100 μl of the complete RPMI-1640 (hereinafter abbreviated as “HAT medium”) containing 10E-4M hypoxanthine, 4.0 × 10E-7M aminopterin, and 1.6 × 10E-5M thymidine is added to each well. Thereafter, half of the supernatant was replaced with fresh H on days 2, 3, 5, 8 and 11 respectively.
The medium was replaced with AT medium, and half of the supernatant was mixed with 1.
0x10E-4M hypoxatin, 1.6x10E-5M
The complete RPMI-1640 containing thymidine (hereinafter abbreviated as "HA medium") is replaced. Similarly, 18, 20, 23
And on day 26, half of the supernatant is replaced with HT medium and on day 28, half of the supernatant is replaced with complete RPMI-1640. Thereafter, the growth and maintenance of this complete RPMI-1640 are carried out. The hybridoma thus obtained was cloned by the limiting dilution method. That is, 3 hybridomas / m
, Balb / c mouse thymocytes 1 × 10 −E7 / m
It was adjusted to complete RPMI so that it would be 0.2 m / well, and the cells were seeded in a 96-well plate and cultured.
The growing hybridoma was further cloned in the same manner. For the search for clones producing the desired antibody, 43 clones of hybridomas were obtained by an ELISA method using a peroxidase-labeled goat anti-mouse immunoglobulin antibody using a 96-well plate coated with immunogen (manufactured by Dynatec Laboratory). (First screening). Furthermore, each monoclonal antibody of 43 selected clones was reacted with human serum containing cholinesterase, followed by reaction with anti-mouse IgG goat IgG, and the immunoprecipitation method for measuring the enzyme activity present in the centrifugal supernatant ( According to FIG. 4), 20 clones were obtained (second screening). Then, immunogen was added to Sephacryl s-300 (2.5 × 9
0 cm), and separated the SCE fraction and cholinesterase of the molecular weight 300,000 to 500,000 large active fraction, and using a 96-well plate (manufactured by Dynatec Laboratories) coated with 7 μg / well of each fraction, As a result of selection by the ELISA method using a goat anti-mouse immunoglobulin antibody labeled with peroxidase (Fig. 5), SC
Nine clones were obtained that reacted only with the E fraction but not with the highly active fraction (third screening). Furthermore, 2 clones (NO. 11, 29) were selected from the above 9 clones. And among the above two clones, the clone NO.S which is particularly excellent
The desired hybridoma designated CE-29 was obtained.

A desired hybridoma represented by NO.SCE-29 was obtained.

3; Preparation of antibody (1) The hybridoma of clone NO.SCE-29 obtained in 2 above was cultured in complete RPMI-1640 medium in a 5% carbon dioxide gas incubator at 37 ° C for 48 hours. The culture solution was centrifuged (3,000 rpm, 10 minutes) to obtain a culture supernatant containing the monoclonal antibody of the present invention.

(2) 1 × 10E6 hybridomas of clone NO.SCE-29 obtained in the above 2 were suspended in 0.5 m of RPMI-1640 medium, and pristane (0.5 m
/ Mouse) was administered to Balb / c mice intraperitoneally. After 10 days, the accumulated ascites was collected and antibody S was collected.
Ascites fluid 4-8 m / mouse containing CE-29 was obtained. The concentration of each of these antibodies was 1 to 10 mg / m.

4; Properties of antibody Immunoglobulin class: Rabbit antibody against various mouse immunoglobulin classes (L
itton.Bionetico.Inc.Kensington.MD20795) and 125
-I labeled protein A was used according to the method of Yeh et al. [Mingyang Yeh et al. Proc.Natl.Acad.Sc
i.USA.Vol.76.No.6.pp.2927-2931 (1979)].

The results are shown in Table 1 below.

5; Antibody Specificity (1) The reaction specificity of the antibody of the present invention was determined by using the following two samples, E
It was performed by the LISA method.

Sample 1: The immunogen obtained in the above 1 was used as Sephacryl S-300 (2.
5 × 90 cm), collect the fractions with a molecular weight of 300,000 to 500,000,
It was adjusted to 7 μg / m.

Sample 2: As in Sample 1, immunogen was added to Sephacryl S-
Attach to 300 (2.5 x 90 cm), collect SCE fractions, 7 μg / m
Adjusted to.

The reactivity of the above-mentioned sample with the monoclonal antibody of the present invention was tested according to the ELISA method in the same manner as in the search for the above-mentioned clone 1. As a result, it was confirmed that the monoclonal antibody of the present invention reacts with SCE.

(2) 1 m of human serum was applied to Sephacryl S-300, and the reactivity of each fraction obtained with this monoclonal antibody was assayed by the EIA method. That is, the sample was applied to the ELISA plate (96-well flat bottom plate) having the monoclonal antibody immobilized thereon at 50 μ / well.
Addition is continued for 2 hours at room temperature. Next, after removing unreacted substances, oxygen-labeled cholinesterase monoclonal antibody is reacted at room temperature for 2 hours. Finally, after removing unreacted substances,
The reaction was carried out by adding a substrate, and the reactivity with this monoclonal antibody was measured from the degree of color development.

As shown in FIG. 1, 90% or more of cholinesterase present in serum has a molecular weight of 300,000 to 500,000, and the remaining 10% or less is SCE. As shown in FIG. 2, this monoclonal antibody was confirmed to react with cholinesterase having a molecular weight of 70,000 to 300,000.

The cholinesterase was measured using Choline E reagent (international reagent).

(3) The results of SCE measurement in the sera of healthy subjects, patients with cirrhosis and patients with fatty liver are shown in FIG. The liver cirrhosis patient shows a low value, and the fatty liver patient shows a high value, as compared with a healthy person.

[Effects of the Invention] Since the present monoclonal antibody specifically reacts with SCE, the use of this antibody provides an immunological measurement technique and an immunological purification technique. If the SCE of the subject is measured, it is possible to diagnose liver disorders such as cirrhosis and fatty liver.

[Brief description of drawings]

FIG. 1 is a molecular weight distribution map of cholinesterase present in human serum using gel filtration, FIG. 2 is a molecular weight distribution map of cholinesterase that reacts with the present monoclonal antibody, and FIG. The figure which shows the measurement result of SCE in the serum of a liver cirrhosis patient and a fatty liver patient, FIG. 4 is the antibody specificity figure by an immunoprecipitation method, and FIG. 5 is the antibody specificity figure by an ELISA method.

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Claims (1)

[Claims] 1. A method of producing cholinesterase produced by fusion cells of immune cells of mammals immunized with cholinesterase and myeloma cells of mammals and reacting with cholinesterase having a molecular weight of 70,000 to 300,000 present in human body fluids. Monoclonal antibody.