JPH02245195A - Monoclonal antibody to specifically react to cholinesterase having 70,000-140,000 molecular weight existing in human plasma - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody useful for immunological purification and measurement of cholinesterase, comprising a monoclonal antibody having specific reactivity to relatively low-molecular cholinesterase existing in human serum. CONSTITUTION:A monoclonal antibody having specific reactivity to cholinesterase having 70,000-140,000 molecular weight existing in human plasma is produced by cell fusion of an immunocyte of mammal immunized against cholinesterase and a mammalian myeloma cell.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to small cholinesterase (hereinafter referred to as r
This invention relates to a monoclonal antibody that has specific reactivity to scEJ.

[Prior art] Cholinesterase (3
, 1, 1, 8) refers to an enzyme that hydrolyzes choline esters into choline and organic acids, but serum cholinesterase is a nonspecific enzyme ( Pseud
o-choli nesterase) and is a glycoprotein containing sialic acid and having a molecular weight of 70,000 to 500,000. So far, the creation of a monoclonal antibody that specifically reacts with cholinesterase with a molecular weight of 300,000 to 500,000 [St
ephenBrimijain, Mo1eculer
Pharmacology, 24:513-520
(1983)].

[Problems to be Solved by the Invention] Serum cholinesterase is produced in the liver and supplied to the serum, and its activity is acutely reduced in the event of liver parenchymal damage. Therefore, its measurement is performed as an important liver function test. It is. In addition, since cholinesterase is present in arteriosclerotic lesions where cholesterol esters are accumulated, it is possible that it acts on accumulated lipids and is involved in lipid metabolism and furthermore arteriosclerosis. It has been suggested that this research is attracting attention.

However, the physiological role of cholinesterase has not yet been fully clarified due to the absence of short-chain fatty acid esters, which are the substrates of cholinesterase, in the blood [Kutty, et al.] 1. , Review, Biolo
logical function of cholines
terase, clin, Biochem, 13
, 239-243 (1980)], it is simply regarded as an activity.

Therefore, there is a need for separation and purification techniques and measurement techniques for examining the properties of cholinesterase in more detail and analyzing its mechanism of action.

Furthermore, even if the conventional monoclonal antibody that specifically reacts with cholinesterase with a molecular weight of 300,000 to 500,000 is known, a monoclonal antibody that specifically reacts with cholinesterase with a molecular weight of less than that is not known. .

The present invention was made from the above-mentioned viewpoint, and an object of the present invention is to provide a monoclonal antibody having specific reactivity to SCE present in human plasma.

[Means and Actions for Solving the Problems] In order to achieve the above objects, the present invention provides cholinesterase-immunized mammalian immune cells and mammalian myeloma cells that are produced by fusion cells, which are produced in human plasma. The gist is a monoclonal antibody characterized by having specific reactivity to existing SCE.

Furthermore, by using the monoclonal antibody obtained according to the present invention, it has become possible to immunologically purify and measure SCE.

The monoclonal antibody of the present invention is produced by cell fusion of a mammalian immune cell immunized with cholinesterase and a mammalian myeloma cell, and its most distinctive feature is that it has specific reactivity to SCE.

The monoclonal antibody of the present invention will be further described in detail below.

Cholinesterase used as an immunogen is one prepared by methods described in the literature [Bio
Chemica et Biophysica Act
A, 570 (1979) 8B-95 etc. 1 and purified products of SCE, any product containing SCE can be used. In addition, the mammal to be immunized with the immunizing antigen does not need to be particularly limited, but is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion.
Generally, mice, rats, etc. are advantageously used.

Immunization is carried out by an intrashell method, for example by administering the above-mentioned immunizing antigen to the mammal by intravenous, subcutaneous, subcutaneous or intraperitoneal injection.

More specifically, the immunogen is administered to the animal several times every 2 to 14 times, optionally in combination with a conventional adjuvant, so that the total dose is about 100 to 10.00 μΩ/mouse. is preferred. As the immune cells, it is preferable to use lung cells extracted about three times after the final administration.

In addition, as the mammalian plasmacytoma cell as the other parent cell to be fused with the above immune cell, various known cell lines such as p3 (p3/X63-Ag8) [N
ature, 256, 495-497 (1975)]
, D3-U 1 [Current Topics
in Microbiology and Immunology
ology, 81.1-7(197&)], N5-
1 [Eur, J.

Immunol, 6, 511-519 (1976)
], MP(, -11[Ce1l, 8.405415(
1976)], 5P-210 [Nature, 27
6.269-270 (1978)], FO [J.

Immunol, ) leTH,, 35, 1-21 (1
980th month, X63.6°5, 3. [J, Immuno
l,, 123.1548-1550 (1979)],
S 194 [J, Exp, Med,, 148,
313-323 (1978)], and R in rats.
210 [Nature, 277.131-133 (
Myeloma cells such as [1979)] are used.

The above-mentioned fusion reaction between immune cells and plasmacytoma cells can be carried out basically using known methods such as Milestein (2011).
5tein) et al. [t4method in Enz
ymology, V.

, 73. pt) 3 (1981)]. More specifically, the fusion reaction is carried out in a conventional nutrient medium, for example in the presence of a fusion promoter. As the fusion promoter, commonly used ones such as polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and if desired, in order to increase the fusion efficiency,
Auxiliary agents such as dimethyl sulfoxide may also be used. The ratio of immune cells to plasmacytoma cells used is the same as in normal methods; for example, the ratio of immune cells to plasmacytoma cells may be about 1 to 10 times. As the medium for the above-mentioned fusion, for example, RPMI-1640 medium, MEM medium, which is used for the proliferation of the above-mentioned plasmacytoma cell line, and various other usual media used for this type of cell culture can be used. Normally, it is best to use serum supplements such as fetal bovine serum (Fe2).

For fusion, predetermined amounts of the immune cells and plasmacytoma cells are thoroughly mixed in the medium and heated to a temperature of about 37°C in advance using a PEG solution, for example, a PEG solution with an average molecular weight.

Approximately 30 to 6,000 OOO to normal medium
This is done by adding and mixing at a concentration of 60 W/V%. Thereafter, desired hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

The resulting desired hybridoma is isolated by culturing it in a normal selection medium, such as HAT medium (a medium containing hyboxanthin, aminopritene, and thymidine). The time required is sufficient for the cells (unfused cells, etc.) to die, usually from several days to several weeks.
A search for a strain producing the antibody of interest and single cloning are performed.

The search for the producing strain can be performed, for example, by the ELISA method [Engva
ll, E,, METH, Enzymol,, 70,
419-439 (1980)], plaque method, spot method, agglutination reaction method,
Various methods commonly used for detecting antibodies, such as the onion method and radioimmunoassay (RIA) method ["Hybridoma method and monoclonal antibodies", published by R&D Planning Co., Ltd., I) I) 30-53. March 5, 1982]. Moreover, this search is performed using the above-mentioned immunizing antigen.

The thus obtained hybridoma producing antibodies that recognize SCE can be subcultured in a normal medium,
It can also be stored for long periods in liquid nitrogen.

The monoclonal antibody of the present invention can be collected from the hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and allowing it to grow, and then collecting the ascites from the hybridoma. The method of obtaining the information will be adopted. The former method is suitable for obtaining highly purified antibodies, and the latter method is suitable for mass production of antibodies. Furthermore, the antibody obtained as described above can be further purified by conventional purification means such as salting out, gel filtration, and affinity chromatography.

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

Purification and measurement of SCE are performed according to conventional methods except for using the monoclonal antibody of the present invention. The method for measuring SCE will be described in detail below.

Measurement of SCE can be performed using known conventional immunoassay methods, such as RI.
It can be carried out according to method A, enzyme immunoassay (EIA), etc., and the operations and procedures for each of these immunoassays are not particularly different from those generally employed, such as the known direct method, indirect method, competitive method, etc. Act, Sanderutsch Act, etc. can be applied.

For diagnostic purposes, cells, tissue pieces, and/or various body fluids are used as specimens in the above measurement method.

When cells and/or tissue pieces are used as specimens, conventional direct or indirect immunization methods are used. According to this method, physiological saline or normal phosphate buffer (PB
The monoclonal antibody of the present invention is immunoreacted with cells suspended in a buffer such as S) or with cells or tissue sections fixed on a glass slide, etc., and the cells or tissue sections are thoroughly washed with the above buffer. The presence of the monoclonal antibody of the present invention bound to cells or tissue pieces may be directly or indirectly assayed in the usual manner. Some of the labeling agents for the above-mentioned assay will be described later, but ordinary fluorescence, enzymes, etc. can be used.

Conventional methods can also be followed when using body fluids as the measurement material. Here, as the body fluid, for example, blood, cell tissue fluid, lymph fluid, pleural fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, cerebrospinal fluid, saliva, etc., or centrifuged supernatant after solubilizing the above cells or tissue pieces, etc. are used. can do.

Insolubilization method (method using insolubilized antigen or insolubilized antibody)
When employing the method, the above-mentioned SCE or the monoclonal antibody of the present invention is produced by chemically or physically reacting with an insoluble carrier according to a conventional method. Examples of the insoluble 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, etc. Polymers, amino acid copolymers, ethylene-maleic acid copolymers, etc. can be used. Insolubilization can be carried out using 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 polysaccharification method, cellulose carbide method) nate derivative method, method using a condensation reagent, etc.>, alkylation method, carrier binding method using a crosslinking reagent (using geltaraldehyde, hexamethylene isocyanate, etc. as a crosslinking reagent), UQi
This is carried out by a chemical reaction such as a carrier bonding method by reaction, an ion bonding method using a carrier such as an ion exchange resin, or a physical adsorption method using a porous glass such as glass beads as a carrier.

As the labeled antigen or labeled antibody, those labeled with the above-mentioned SCE or the monoclonal antibody of the present invention with various labeling agents such as conventional radioactive substances, enzyme labeling substances, fluorescent substances, etc. are used. The radioactive substance used as this labeling agent is 125
-I and other radioactive iodine, fluorescent substances such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC).
, substituted rhodamine isothiocyanate (XRITC)
, rhodamine B isothiocyanate, dichlorotriazine fluorescein (DTAF), etc.; enzyme labeling substances include peroxidase (POX), alkaline phosphatase, β-galactosidase, microperoxidase, chymotrypsinogen, procarboxypeptidase, glycero Aldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-nase, R-
Naze, etc. can be mentioned respectively. These labeling methods can also follow conventional methods [J, Bio
l, Chem, 254.9349-9351 (1
979); Nature, 194, 495 (1962
); Fluorescent antibody method, medical chemistry experiment course Nc4, 263-27
0; Acta, Endocrinol, 5uppl
.

168.206 (1972); Proc, Nat.
Acad, Sci, USA.

57, 713 (1967), etc.].

The method of measuring and quantifying the test substance SCE in a specimen according to the present invention is carried out by immunoreacting using any one of the above-mentioned insolubilized antigen, insolubilized antibody, labeled antigen, and labeled antibody. In this case, the solvent used in the measurement system is a usual one that does not have a negative effect on the reaction, such as citrate buffer, phosphate buffer, Tris-HCl buffer, borate buffer,
Preferred examples include buffers having a pH of about 4 to 8, such as acetic acid buffers. In addition, the immunoreaction conditions for the measurement are not particularly limited and can be the same as those for this type of measurement method. That is, the immune reaction is generally carried out at a temperature of 1 to 40°C or less, preferably about 4 to 40°C.
It takes time.

Separation of the conjugate and free form (B-F) after the completion of the immune reaction also follows known methods; for example, when an insolubilization method is adopted,
The solid phase and liquid phase can be separated by separation means such as centrifugation, filtration, washing, and decantation. In other cases, conventional methods such as the dextran-activated charcoal method and the second antibody method may be followed.

A particularly convenient way to carry out the above assay is through the use of kits. 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 the antibody reagent. Preferably, the antibody reagent is lyophilized, and the kit can include a water-soluble or water-miscible solvent. Furthermore, this antibody reagent contains
Buffers can be added to keep the reconstituted reagent system at a constant l1l-() and/or preservatives and/or stabilizers can be added to prevent the sample from deteriorating before use. Although it is not considered an essential component of the kit reagent, when carrying out the above measurement method, it is preferable to use one with p'r-1 of about 4 m8.Also, the reconstitution agent preferably contains water. However, part or all of the water can also be replaced by a water-miscible solvent.Water-miscible solvents are well known to those skilled in the art, such as glycerin,
Alcohols, glycols, glycol ethers, etc. can be used.

[Example] 1; Purified immunogen of fresh human serum 200d was added to 10mM potassium phosphate buffer (
Dialysis was performed for 24 hours at below 4°C against pH 8.0>.

Next, this was equilibrated with the same buffer solution.
ammonium anilinium 230mg
Coupled 5epharO3e column (2,5X 10c
After washing with the same buffer 20 (M!),
The target enzyme was eluted with the same buffer containing M NaCL. This was mixed with 10Tr1.1 containing 50mM NaCL. After dialysis with M phosphate buffer, Trimethylam
monium ani l inium SephaO
3e column and 200mf of the same buffer! After washing with O
A concentration gradient was applied from TrLM to 200 mM NaCL to separate the fraction containing the target enzyme. Furthermore, this is 10m
After dialysis with M potassium phosphate buffer, it was equilibrated with the same buffer and applied to a DEAE-3 ephadex column (1,2 x 16
c> from 10mM potassium phosphate buffer to 200%
A concentration gradient was applied to TrLM phosphate buffer to obtain 0.5 m9 of cholinesterase containing SCE. After dialysis and concentration, it was stored at below 20°C.

2; Hybridoma production Immunogen and Freund's combined Aschband at 1:1
250 μL (20 μg of immunogen) was mixed with Ba
I b/c mice (female, 8 weeks old) were injected sarcastically into the back. Furthermore, the same amount of immunization was performed a total of 6 times at 2-week intervals. Three days after the final immunization, the lungs were removed and the tile cells were treated with RPMI-1640.
Wash 3 times with medium. After washing the mouse myeloid cell line N5-1 in the same manner, 7 cells of this NS-"l, 2.2X10E,
Eight 1°6×10E cells were placed in a 50° centrifuge tube and mixed.

After centrifuging at 200XQ for 5 minutes, remove the supernatant with a Pasteur pipette. Polyethylene glycol 4000 (Sigma >50w/v% RPMI-1 kept at 37°C)
640 solution 0. ! Add M dropwise over 1 minute and mix slowly for 7 minutes. 15%FC3.1Tr kept at 37°C
LM Pyruvate RPMI-1640 (hereinafter [Complete RP
(abbreviated as MI-1640J) was added for 1 minute, then the same amount of complete RPMI-1640 was added for 1 minute, then 8
Add the complete RPM 1 dropwise and stir slowly for 2 minutes.

After centrifugation at 200XCI for 5 minutes, remove the supernatant, suspend in complete RPMI-1640 kept warm at 37°C at 1 x 10E7 cells/i, and inoculate Microtest-■ plates (manufactured by Falcon) at 100μC each. and 37°C15%
Culture in a carbon dioxide incubator. 24 hours later 1
．． Ox10E-4M Hyboxanthin, 4. OXloE-
Complete RPMI-1640 (hereinafter referred to as rHAT medium) containing 7M aminopterin, 1.6X10E-5M thymidine
Add >100 μL, abbreviated as , to each well. Thereafter, half of the supernatant was replaced with fresh HAT medium on days 2.3.5.8 and 11, respectively, and half of the supernatant was replaced with fresh HAT medium on day 14. OXloE-4M Hypoxanthine, 1.6x10
Complete RPMI-'1640 containing E-5M thymidine (
(hereinafter abbreviated as "It-IA medium"). Similarly, on days 18.20.23 and 26, half of the supernatant was replaced with H-cho medium, and on day 288, half of the supernatant was replaced with complete RPMI-16.
Change it to 40. Thereafter, the cells are grown and maintained using this complete RPMI-1640. The hybridoma thus obtained was cloned by the limiting dilution method. That is, 3 hybridomas/mj! , Ba I b/C'? Mouse thymocyte lX1O-E7/mj! Adjusted to complete RB PMI so that this 0.2m! ! The cells were plated and cultured in a 96-well plate so that the number of cells per well was obtained. The proliferating hybridomas were further cloned in the same manner. To search for clones that produce the desired antibody, use 5 epha of immunogen.
Attach to cryl S-300 (2.5x90cm),
CE and cholinesterase with a molecular weight of 300,000 to 500,000 were separated, and 96-well plate coated with 7 μg/well of each fraction (manufactured by Dynatic Laboratory)
The test was carried out by ELISA using a peroxidase-labeled goat anti-mouse immunoglobulin antibody. In this way, the desired hybridoma represented by clone No. 5CE-5 was obtained.

3: Preparation of Antibodies (1) Each hybridoma, clone No. 5CE-5 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) Six 1×10E hybridomas of clone No. and 5CE-5 obtained in 2 above were suspended in 0.5 d of RPMI-1640 medium, and pristane (0,!M/
(mouse) was administered intraperitoneally to 3alb/C mice. After 10 days, the accumulated ascites was collected and treated with antibody 5CE.
4-8 m/mouse of ascites containing -5 were obtained. The concentration of these antibodies is 1-10m! It was j/rd.

4; Antibody properties Immunoglobulin class: Rabbit antibodies against various mouse immunoglobulin classes (
Lotton, Bionetico, Inc.
ensington.

MD20795) and 125-I labeled protein A according to the method of Yeh et al. [Mtng-
YangYeh et al., Proc., Natl.
, Acad, Sci, USA.

VOl, 76, No. 6, pp, 2927-2931
(1979)].

The results are shown in Table 1 below.

Table 1 5; Antibody specificity (1) ELISA method The reaction specificity of the antibody of the present invention was evaluated using the following two samples.
This was done using the LISA method.

Sample 1: The immunogen obtained in 1 above was treated with 5ephacry
IS-300 (2,5X90Cm), molecular weight 3
Fractions from 00,000 to 500,000 were collected and adjusted to 7 μg/d.

Sample 2: Same as sample 1, immunogen was added to Sepha.
cm attached to S-300 (2.5x90cm), SCE
Fractions were collected and adjusted to 7 μg/weight.

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

(2) Affinity serum 172 was combined with the antibody of the present invention 5epharose 4
B column (1, IX3Cm) to obtain a flow-through fraction and an elution fraction with an ammonia solution (pH 11>).These were each concentrated to 1d, and 5ephacry+ 3-30
0 (1.5 x 100 cm). As shown in Figure 1, approximately 90% of phosphoesterase present in serum has a molecular weight of 300,000 to 500,000, and the remaining 10% is SCE.
There was no SCE in the flow-through fraction of the 5epharose 4B column bound to the antibody of the present invention (Fig. 2), and there was no SCE in the eluted fraction.
E exists (Figure 3).

Therefore, it was confirmed that the antibody of the present invention reacts with SCE. Note that cholinesterase was measured using choline E reagent (International Reagent).

[Effects of the Invention] Since the monoclonal antibody of the present invention specifically reacts with SCE, by using this monoclonal antibody, for example, by applying affinity chromatography, etc., an immunological purification technique for monoclonal antibodies can be provided. Furthermore, an immunological measurement technique for this substance is provided.

[Brief explanation of drawings]

Figure 1 is a distribution map of the molecular weight of cholinesterase present in human serum using gel filtration method, Figure 2 is a distribution diagram of the molecular weight of cholinesterase that does not react with the monoclonal antibody of the present invention, and Figure 3 is a distribution diagram of the molecular weight of cholinesterase present in human serum. This is a distribution map of the molecular weight of cholinesterase that reacts with monoclonal antibodies. March 20, 1989 Procedural Neho (Spontaneous) March 27, 1990

Claims (1)

[Claims] 1 Produced by a fusion cell of a mammalian immune cell immunized with cholinesterase and a mammalian myeloid cell,
A monoclonal antibody characterized by having specific reactivity with cholinesterase having a molecular weight of 70,000 to 140,000, which is present in human plasma.