JPS63240797A - Monoclonal antibody, production thereof and diagnostic containing said antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody specifically bonded to membrane protein coded from human chromosomes having abnormal numbers. USE:A diagnostic for chromosomal aberration symptom, Down's syndrome, Alzheimer's syndrome, etc. PREPARATION:For example, the 21st. human chromosome is introduced and suspension of Chinese hamster ovarian cell having a membrane protein coded from the above-mentioned chromosome is administered to abdominal cavity of BALB/C mouse to immunize the mouse and after final immunization, the spleen is extirpated to provide an antibody-producing cell, which is then fused with a myeloma cell and the resultant fused cell is cultivated in HAT culture medium to afford a hybridoma, which is further selected and subjected to screening and cloning in order to give monocloning and the resultant hybridoma is cultivated to provide the aimed monoclonal antibody.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a monoclonal antibody that specifically binds to a membrane protein encoded by a human chromosome with an abnormal number, a method for producing the same, and a chromosomal abnormality containing the antibody. This relates to a diagnostic agent.

[Conventional technology]

In recent years, genetic diseases and non-inherited chromosomal congenital anomalies have been on the rise, and this trend has become a worldwide phenomenon. Until recently, medical elucidation of these abnormalities has mainly focused on analysis of external factors, and analysis and testing methods for genetic factors, particularly chromosomal abnormalities, have been extremely limited. This is because chromosomes cannot be observed in normal cells, but only in cells undergoing division, and special techniques were required to fix them. Therefore, early diagnosis of chromosomal abnormalities, especially prenatal diagnosis, is a social demand, and there is a need for the establishment of easy and reliable diagnostic methods.

Fertilized eggs with chromosomal abnormalities are often naturally selected early in pregnancy, but about 1% of babies are born with abnormalities, and many of them have malformations or severe mental developmental disorders. Types of chromosomal abnormalities are broadly divided into numerical abnormalities and structural abnormalities.Triploid, where one egg is fertilized by two sperm, does not grow even after birth, whereas the same chromosome does not grow in the nucleus. People with trisomy (having an excess of 47 chromosomes in the nucleus, usually 46) grow up with mental and physical abnormalities, so society as a whole needs to take action. has been done. For example, Down syndrome (hereinafter simply referred to as "Down's disease") is caused by having one extra chromosome 21 (21-lisomy). As a result, they tend to show high values, and a method for early prenatal diagnosis is desired.

Furthermore, in recent years, while the average lifespan of humans has increased, the number of patients with Alzheimer's disease, so-called senile dementia, has increased, and this is becoming a major social problem.

Although Alzheimer's disease usually develops after the age of 60, early diagnosis methods are desired from the viewpoint of prevention and treatment. Conventionally, antemortem diagnosis of Alzheimer's disease has been made by a combination of psychological tests and brain imaging findings (CT, PET, etc.), but although it requires complex and expensive equipment, it has not been possible to diagnose Alzheimer's disease after death using brain autopsy findings. It has a problem of lower reliability compared to other methods. Therefore, it is desired to establish a simple and reliable antemortem diagnostic method.

Since the aforementioned Down's disease shows symptoms similar to Alzheimer's disease at around the age of 25, there has been interest in the genetic relationship between the two, but recently it has been discovered that some Alzheimer's patients also have chromosome 21. An abnormality in the number of numbers was reported (J, M.

Delabar et al.
Genetique, 2! Me, 226
-228 (1987)).

[Purpose of the invention]

Therefore, the object of the present invention is to provide a novel monoclonal antibody that is useful for easily and reliably diagnosing various diseases caused by an abnormal number of chromosomes, a method for producing the same, and a diagnostic agent containing the monoclonal antibody. It is about providing.

The present inventors have already applied known cell fusion technology to successfully obtain a monoclonal antibody that can detect numerical abnormalities in human chromosomes with high sensitivity. Furthermore, the use of this monoclonal antibody led to the development of a diagnostic agent useful for diagnosing chromosomal abnormalities, particularly for diagnosing Down's disease or Alzheimer's disease.

[Structure of the invention]

That is, the present invention provides a monoclonal antibody that specifically binds to a membrane protein encoded by an abnormal number of human chromosomes.

The monoclonal antibody of the present invention can be obtained by, for example, immunizing an animal with Chinese hamster ovary cells into which a human chromosome has been introduced and which has a membrane protein encoded by the human chromosome, and converting the antibody-producing cells obtained from the animal into myeloma cells. fuse to form hybridomas, select
By screening and cloning, a hybridoma that produces a monoclonal antibody that specifically binds to a membrane protein encoded by an abnormal human chromosome is obtained,
It can be obtained by a method comprising various steps of culturing the hybridoma.

Chinese hamster ovary cells (CHO cells) into which human chromosomes used for immunization have been introduced using the above production method and which have membrane proteins encoded by the human chromosomes.
can be obtained by known cell fusion techniques. An example of such a CHO cell is a CHO cell in which human chromosome 21 has been introduced and has a membrane protein encoded by the chromosome.
Some HO cells are called 2Fu' (D, Patterson et al., Som, Ce1l G
enet, supra, 91-110 (1975))
.

When immunized with 2Fu', chromosome 2
Monoclonal antibodies can be produced that can detect trisomy 21, which is a number abnormality in trisomy 1. Other known abnormalities in the number of chromosomes include trisomy 18, trisomy 13, Talainefelter syndrome, triple X syndrome, and Turner syndrome, but monoclonal Antibodies can also be produced by using CHO cells into which a corresponding human chromosome has been introduced and which has a membrane protein encoded by the chromosome.

The animals to be immunized used in the above method include, for example,
Examples include mice and rats, but mice are usually used.

The route and schedule of administering the antigen to the animal can vary. For example, in a preferred mode of administration, the antigen is treated with mitomycin and then injected intraperitoneally in several doses. Antibodies are produced in the animal's lungs upon administration of the antigen.

Lung cells are usually used as antibody-producing cells.

Next, as myeloma cells to be fused with the obtained antibody-producing cells, for example, BALB/C mouse MOPC2
Cells derived from 1 myeloma (referred to as NS-1) are used. This K5-1 is resistant to 8-azaguanine and dies in a medium containing hypoxanthine-aminopterin-thymidine (HAT medium) because it lacks the enzyme hypoxanthine-guanine-phosphoribosyltransferase. , which is convenient for selective culturing of the resulting hybridomas. Polyethylene glycol is usually used as a fusion agent for cell fusion.

The above fusion technique and myeloma cells used are known per se [myeloma cells;
al, +Fur, J, Immuno1. +6.
292-295 (1976). Fusion method; K16
Hler et al., ibid., 6.511-516.
(1976)).

Hybridomas are then screened according to conventional methods and then cloned to obtain individual hybridoma progeny from each line.

This cell line, that is, the clone, is grown in a cell culture medium (in vitro culture).
Alternatively, monoclonal antibodies that specifically bind to membrane proteins encoded by an abnormal number of human chromosomes can be synthesized and grown indefinitely in vitro (in vivo culture).
Continue to secrete. The monoclonal antibodies produced are recovered by methods known in the art.

For example, when 2Fu' is used as an antigen in the above production method, the monoclonal antibody obtained in this way is specific to a membrane protein encoded by chromosome 21, which has an abnormal number, when targeting fibroblast cells. It is something that connects to each other.

The present invention also provides a diagnostic agent containing the above-mentioned monoclonal antibody. When this diagnostic agent is applied to, for example, human fibroblast cells, abnormalities in the number of chromosomes can be easily determined. Therefore, for example, if a monoclonal antibody specifically binds to a membrane protein encoded by abnormal chromosome 21, Down's disease and Alzheimer's disease can be easily diagnosed. The diagnostic agent of the present invention also includes:
It can be prepared for diagnosis of 18-trisomy, 13-trisomy, Talainefelter syndrome, Triple X syndrome, Turner syndrome, etc.

The diagnostic agent of the present invention may be used in any immunological testing method, such as immunofluorescence, immunoadhesive hemagglutination, radioimmunoassay, and the like. For example, when immunofluorescence is used, human cells are treated with the monoclonal antibody of the present invention, and then a fluorescent dye (
For example, the presence or absence of a chromosomal abnormality can be easily diagnosed by reacting with anti-mouse IgG labeled with FITC, setting it on a fluorescence microscope, and checking the luminescence state.

〔Example〕

The monoclonal antibody and diagnostic agent of the present invention will be explained below with examples.

Example 1 (1) (Production of antibody) Human chromosome 21 has been introduced, and this chromosome 21
CHO cells with membrane proteins encoded by
2Fu') was treated with mitomycin and washed with PBS (phosphate buffer). 5 x 10' cells/0.5 c
A PBS suspension of 2Fu' was injected (immunized) into the peritoneal cavity of female BALB/C mice. 2 weeks later, lX
l0' cells/0.5 cc, after another 2 weeks lXl0'
Cells/0.5 c'c were injected, and 3 days later, the lungs of the same mice were removed, triturated in RPMI-1640 medium, washed three times by centrifugation (1500 rpm), and resuspended in the same medium. It got cloudy.

(2) (Cell fusion) Lung cells obtained above 1. lX10” and RPMI
1.0 x 10' BALB/C mouse myeloma cells (NS-1) washed once with -1640 medium were mixed,
A pellet was prepared by centrifugation at 1500 rpm for 5 minutes.

Next, a solution of polyethylene glycol (1540) dissolved in RPMI-1640 medium to 50 wt% was prepared.
1 liter was added under stirring, and further RPMI-1640 medium was added to the mixture so that the total amount was 10 mj2, and the mixture was stirred. After the end,
After centrifuging at 11,000 rpm for 5 minutes to remove the fusion agent polyethylene glycol, l? PMI-1640
40 mj medium containing 20 vol 1% of fetal bovine serum (Fe2) added to the medium! resuspended in.

(3) (Selection, Screening, Cloning) Add 0.2mj of the above suspension to each of the 192 wells of a 96-well tissue culture plate.
! (cell number 5 x 10b), and add half of the culture supernatant every 2 to 3 days to HAT medium (hypoxanthine 136.1
■/II+7! , aminopterin 1.76■/ral,
Substitution was made with 38.75 ml of thymidine. The culture conditions were 37° C., 100% humidity, and CO□ gas concentration of 7%. After 10 days, hyperdomas were observed in 119 wells (occurrence rate 62%). The supernatant of the 119 wells that showed a positive result was
When Fu' was screened to see if antibodies were produced by enzyme-linked immunosorbent assay (ELISA), 20 holes were positive (10.6% of gold holes).

BALB/C mouse thymocytes 1
．． Limit dilution was carried out with HAT medium supplemented with 0x10' cells/ml, and 0.2 ml was placed in each hole of a 96-well tissue culture plate and cultured. At this time, the number of hybridomas per hole and the number of holes are 48 holes for 1 cell/hole, 45 holes for 5 cells/hole, and 20 holes for 5 cells/hole.
There were 3 cells/holes. The culture conditions were the same as above.

The supernatant was subjected to CH
As a result of screening for O cells minus and human leukemia cells (TALL) plus, clones were obtained in 3 wells.

Furthermore, the three remaining large cells were re-loned twice using the limiting dilution method, and each of the resulting hybridomas was
They were named K-1, 0K-2, and 0K-3.

Monoclonal antibodies produced from hybridomas 0K-1, 0K-2 and 0K-3 were examined by enzyme immunoassay using anti-mouse antibodies, and were found to be of class rgM, respectively.
It turned out to be rgG and IgM.

The following proteins are recognized and bound by the monoclonal antibodies produced by 0K-1, 0K-2 and 0K-3.

・0K-1-producing monoclonal antibody: Recognizes four bands in Western blotting of human leukemia cell (TALL) protein.

・0K-2-producing monoclonal antibody: Recognizes 40 and 90 kilodalton bands in Western blotting of TALL cell proteins.

- 0K-3-producing monoclonal antibody: Recognizes four bands of 86.4, 74.2, 61.4, and 3664 kilodaltons in Western blotting of TALL cell proteins. In Western blotting of 2Fu' cell protein, 89.8
Three bands of 2 and 45 kilodaltons are recognized.

Example 2: Diagnosis of fibroblasts from normal individuals or patients with Down's disease using the method
A microscope cover glass (12X12mm
) and cultured each cell. As a medium, MEM was supplemented with non-essential amino acid (NEA).
A) A mixture containing 10% sodium pyruvate, 10mM lactalbumin hydrolysate (LAH) and 0.1% FC3 was used. After incubation at room temperature, coverslips were washed 2-3 times with PBS, immediately fixed in 3.7% formaldehyde in PBS, washed with cell distilled water, and further fixed in acetone at -20°C.

15 μl of the monoclonal antibody obtained in Example 1 (derived from hybridomas 0K-1, 0K-2, or 0K-3) was applied to the fixed cells and allowed to react at room temperature for 1 hour. P.B.
After washing three times with S, a second antibody, FITC-labeled anti-mouse IgG antibody or anti-mouse IgM antibody, was added once
5 μl was added and allowed to react at room temperature for 1 hour. The specimen was washed three times with PBS and once with distilled water to remove salt, placed on a glass slide with the side on which the cells were fixed facing down, and sealed with gelbatol to obtain a piece of the specimen.

The specimen was examined using a fluorescence microscope to determine whether or not the surface layer emitted light. Table 1 shows the results obtained. The fibroblasts of Down's disease patients are fluorescent, whereas those of normal people do not emit light.
It can be seen that the presence or absence of chromosomal abnormalities can be diagnosed easily and with high sensitivity without using special techniques.

In addition, in order to confirm that proteins were present on the membrane at the same time as this method, we also searched for membrane immunofluorescence, in which cells are not fixed and reacted with a fluorescent dye, but the results were the same as above. Met.

Table 1 Presence or absence of luminescence by fluorescence Example 3 Fibroblasts from normal people and Alzheimer's disease patients were cultured using the same medium used for fibroblasts in Example 2. The test was conducted in the same manner as in Example 2. The results are shown in Table 2.

Table 2.1: No older brother [Effects of the invention] The monoclonal antibody of the present invention specifically binds to a protein encoded by an abnormal human chromosome.
For example, it is useful for diagnosing disorders caused by abnormalities in the number of chromosomes, such as Down's disease and Alzheimer's disease.

Claims (7)

[Claims] (1) A monoclonal antibody that specifically binds to a membrane protein encoded by an abnormal number of human chromosomes. (2) The monoclonal antibody according to claim 1, wherein the number of abnormal human chromosomes is human chromosome 21. (3) Immunize an animal with Chinese hamster ovary cells into which human chromosomes have been introduced and which have membrane proteins encoded by the human chromosomes, and form hybridomas by fusing the antibody-producing cells obtained from the animal with myeloma cells. A method for producing a monoclonal antibody, comprising the steps of: obtaining a hybridoma that produces a monoclonal antibody that specifically binds to a membrane protein encoded by an abnormal human chromosome by selection, screening, and cloning, and culturing the hybridoma. (4) A method for producing a monoclonal antibody according to claim 3, wherein each of the human chromosomes is human chromosome 21. (5) A diagnostic agent for chromosomal abnormalities containing a monoclonal antibody that specifically binds to a membrane protein encoded by a human chromosome with an abnormal number. (6) The diagnostic agent for Down's disease according to claim 5, wherein the number of abnormal human chromosomes is human chromosome 21. (7) The diagnostic agent for Alzheimer's disease according to claim 5, wherein the abnormal human chromosome of the above number is human chromosome 21.