Note: Descriptions are shown in the official language in which they were submitted.
1288~7~L
Monoclonal antibodY, process for Preparation thereof and
diagnostic drug containinq the same
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to a monoclonal antibody
which specifically binds to a me~brane protein encoded
by human chromosome No. 21 having aberration in number, a
process for preparation thereof, and a diagnostic drug
for Down's syndrome containing the antibody.
2. Description of the prior art
Recently, hereditary and nonhereditary congenital
chromosomal aberration diseases have been increased, and
this tendency has become a worldwide phenomenon. Medical
approaches against these aberration diseases mainly
depend on analysis of exanthrope up to recently, and
analysis and test method thereof on hereditary factors,
particularly chromosomal aberration have been extremely
limited. This is because since chromosome cannot be
observed in ordinal cells and can only be observed in
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cells in mitotic period, special techniques are required
for fixing method thereof. Therefore, early diagnosis,
particularly prenatal diagnosis of chromosomal
aberrations is a social demand, and establishment of easy
and highly confidential diagnostic methods therefor has
been desired.
Though many of fertilized eggs having any chromosomal
aberration are naturally selected during early phase of
pregnancy, about one percent of born children are born in
a state having the aberration, and thus many of them have
malformations and/or intensive disturbances in anima
development. Kind of chromosomal aberration is
classified into aberration in number and aberration in
lS structure. Triploid formed by fertilizing one egg with
two sperms does not grow even if it is born, whereas
since a patient of trisomy wherein the same chromosome
excessively exists in the nucleus grows accompanying
mental and physical aberrations, wrestling therewith from
whole society has been desired. 21-Trisomy having the
chromosome No. 21 in an excess number of one causes
Down's ~yndrome (hereinafter simply referred to as
"Down's disease"). Natality of children of Down's
disease tends to increase in accordance with recent
increase of marriage in high age, and thus methods for
early diagnosis prior to childbirth have been desired.
SUMMARY OF THE INVENTI~N
It is a feature of one embodiment of the present invention to
provide novel monoclonal antibodies useful for diagnosing, in
simple and high confidence, Down's disease caused by
chromosome No. 21 having aberration in number, a process
fcr preparation thereof, and a diagnostic drug containing
said monoclonal antibody.
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The present inventors have succeeded in obtaining mono-
clonal antibodies capable of recognizing in a high sensitivity
the aberration of human chromosome No. 21 in number by apply-
ing known cell fusion techniques. Furthermore, they have
developed a diagnostic drug useful for diagnosis of Down's
disease by utilization of the monoclonal antibodies.
In accordance with an embodiment of the present invention
there is provided a monoclonal antibody that has been produced
by a hybridoma obtained by a process comprising the steps of
immunizing an animal with a Chinese hamster ovary cell into
which human chromosome No. 21 has been introduced and which
has a membrane protein encoded by human chromosome No. 21 and
fusing an antibody-producing cell taken from said animal with
a myeloma cell to form hybridomas, that specifically binds
lS said Chinese hamster ovary cell and that binds a human fibro-
blast cell that is trisomic for human chromosome No. 21 but
does not bind a normal human fibroblast cell.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a monoclonal antibody which can
specifically bind to a membrane protein encoded by human
chromosome No. 21 having aberration in number.
It has been discovered that at least one membrane pro-
tein, among the many such proteins encoded by genes located
on human chromosome No. 21, is produced by normal human
fibroblasts, in substantially lesser amounts than by human
fibroblasts containing an aberrant number of chromosome No.
21, as is the case for Down's disease patients. It has also
been found that, in accordance with the present invention,
monoclonal antibodies can be reproducibly obtained that
recognize chromosome 21-polysomic human fibroblast cells by
virtue of binding a membrane protein that is present, at
detectable levels, in such polysomic fibroblast cells but not
in normal fibroblast cells.
Monoclonal antibodies of the present invention are useful
for diagnosis of Down's disease caused by 21-trisomy because
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they speci~ically bind to the membrane protein encoded by
human chromosome No. 21 having aberration in number.
A monoclonal antibody of the present invention can be
obt:ained, for example, by a process comprising steps of
immunizing an animal with a Chinese hamster ovary cell into
which human chromosome No. 21 has been introduced and which
has a membrane protein encoded by the human chromosome No. 21;
fusing antibody-producing cells taken out from said animal
with a myeloma cell to form hybridomas; subjecting the hybri-
lo domas to selection, screening and cloning to obtain a hybri-
doma capable of producing a monoclonal antibody which speci-
fically binds to the membrane protein encoded by human chro-
mosome No. 21 having aberration in number; and culturing the
hybridoma.
The Chinese hamster ovary cell (hereinafter referred to
as CHO cell) into which human chromosome No. 21 has been
introduced and which has a membrane protein encoded by human
chromosome No. 21 used in the above process may be obtained
by known cell fusion techniques. An example of such CH0 cell
includes so-called 2Fur [see D. Patterson et al., Som. Cell
Genet., 1, 91-110 (1975)]. Sample cultures of 2Fur cell are
available from FRI (Fermentation Research Institute Agency of
Industrial Science and Technology), Ibaraki, Japan under the
deposit No. FERM BP-2492.
Examples of the animals to be immunized as used in the
above process include mouse, rat and the like, and mouse is
usually used.
Route and schedule of administration of the antigen into
the animal may variously be varied. For example, according
to a preferred administration method, the antigen is first
treated with mitomycin and intraperitoneally administered by
several portions to the animal. Antibodies are produced in
the spleen of the animal by administration of the antigen.
As antibody-producing cells, spleen cells are usually used.
As the myeloma cell to be fused with the thus obtained
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antibody-producing cells, a cell induced from BALB/C mouse
MOPC 21 myeloma (as referred to as NS-l) is, for example,
used. This NS-l has resistance to 8-azaguanine, and dies in
a medium containing hypoxanthine-aminopterinthymidine (HAT
medium) since it lacks the enzyme hypoxanthine guanine phos-
phoribosyltransferase. Therefore, use of NS-l is convenient
for selective culture of hybridomas to be obtained. As a
fusing agent for cell fusion, polyethylene glycol is usually
used. The above-mentioned cell fusion techniques, myeloma
lo cells to be used, and the like are known, per se [myeloma
cells; Kohler et al., Fur. J. Immunol., 6, 292-295 (1976), and
fusing method, Kohler et al., ibid., 6, 511-516 (1976)].
Resulting hybridomas are in a conventional manner
screened for an antibody-producing ability, and then
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cloned, whereby progenies of respective hybridomas from
respective phyletic lines are obtained. Monoclonal
antibodies which specifically bind to the membrane
protein encoded by human chromosome No. 21 having
aberration in number can successively be synthesized and
secreted by respectively infinitely growing these cell
lines, i.e. clones in a cell culture medium (in vitro
culture) or in uivo. Each of monoclonal antibodies thus
produced can be recovered according to a method known in
the art.
The monoclonal antibody thus obtained specifically binds,
when a fibroblast cell is targeted, for example, to the
membrane protein encoded by chromosome No. 21 having
aberration in number.
The present invention also provides a diagnostic drug for
Down's disease owing to 21-trisomy containing the
above-mentioned monoclonal antibody. Aberration of
chromosome No. 21 in number can readily be determined by
applying this diagnostic drug, for example, to human
fibroblasts or the like. Therefore, diagnosis of Down's
disease can readily be carried out by use of this
diagnostic drug. The diagnostic drug of the present
invention can practically be used through any
immunological test methods, for example,
immunofluorescence, an immunoadhesionhemocyte-
agglutinating reaction method, radioimmunoassay or the
like. For example, in utilization of immunofluorescence,
human cells are treated with the monoclonal antibody of
the present invention, then reacted with an anti-mouse
IgG labeled with a fluorescent dye such as FITC, and set
in a fluorescence microscope, and the state of
luminescence is checked, whereby presence or absence of
chromosomal aberration can readily be diagnosed.
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Monoclonal antibodies and diagnostic drug of the present
i~vention are further explained below according to
non-restricting examples.
Examples
Example 1
(1) Production of antibodies
CHO cells (2Fur) into which human chromosome No. 21 had
been introduced and which had a me~brane protein
encoded by the chromosome No. 21 was treated with
mitomycin C, and washed with PBS ~phosphate buffer). A
2Fur suspension of 5 x 106 cells/0.5 cc in PBS was
intraperitoneally injected into a female BALB/C mouse
(immuni~ation). Two weeks thereafter, a 2Fur suspension
of 1 x 10 cells/0.5 cc in PBS, and further two weeks
thereafter, a 2Fur suspension of 1 x 107 cells/0.5 cc in
PBS were injected in the same manner as above. Three
days thereafter, the spleen of the mouse was taken out,
finely pulverized in RPMI-1640 medium, subjected three
times to centrifugation (1500 rpm) for washing, and
resuspended in the medium.
(2) Cell fusion
1.1 x 108 cells of the spleen cell obtained in the above
procedure and 1.0 x 10 cells of BALB/C mouse myeloma
cell (NS-l) once washed in advance with RPMI-1640 medium
were mixed, and centrifuged at 1500 rpm for 5 minutes to
prepare a pellet. Then, 1 ml of a solution obtained by
dissolving polyethylene glycol (1540) in RPMI-1640 medium
to 50 wt~ was added to the pellet with stirring,
RPMI-1640 medium was added thereto to the total volume of
10 ml, and the mixture was stirred for 6 minutes. The
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mi~ture was subjected to centrifugation at 1000 rpm for 5
minutes to remove polyethylene glycol, and the solid
matters were resuspended in 40 ml of RPMI-1640 medium
containing 20 vol% of fetal calf serum (FCS).
(3) Selection, screening and cloning
0.2 ml (5 x 106 cells) of the above suspension was placed
in each of 192 wells of a 96 wells-plate for tissue
culture, and half amount of the culture supernatant was
replaced every 3 to 4 days with HAT medium (136.1 mg/ml
hypoxanthine, 1.76 mg/ml aminopterine and 38.75 mg/ml
thymidine). The culturing condition was a temperature of
37C, a humidity of 100% and a CO2 gas concentration of
7%. 10 days after the start of the culturing, hybridomas
were observed in 119 wells (emergence rate: 62~).
Culture supernatants in the 119 wells, which revealed
positive results, were subjected to screening by checking
according to enzyme-labeled antibody technique (ELISA)
20 _using 2Fur whether or not the supernatants contain~
antibodies, and the results revealed 20 wells to be
positive (10.6% of the total wells).
Cells in each of the 20 wells were subjected to limiting
dilution using HAT medium with supplementation of BALB/C
mouse thymocyte of 1.0 x 107 cellstml, 0.2 ml portions of
the resulting cell suspensions were placed in each well
of a 96 wells-plate for tissue culture, and culturing was
carried out. In this connection, hybridoma number per
well and well number were 48 wells of 1 cell/well, 45
wells of 5 cells/well and 3 cells of 20 cells/well.
Culturing conditions were the same as above. The culture
supernatants were respectively subjected to the same
ELISA as above-mentioned to screen those which are
negative for a normal CHO cell and positive for 2Fur
and as the result desired clones were
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obtained from three wells. The cells in each of the three
wells were recloned two times according to the limiting
dilution method, and the resulting hybridomas were res-
pectively named OK-l, OK-2 and OK-3. Sample cultures of
hybridomas OK-2 and OK-3 are available from FRI (Fermentation
Research Institute Agency of Industrial Science and Technol-
ogy), Ibraki, Japan under the deposit Nos. FERM BP-1802 and
FERM BP-1803, respectively.
It has been found by an enzyme-labelled antibody tech-
nique using an anti-mouse antibody that monoclonal antibodies
produced from hybridomas OK-l, OK-2 and OK-3 belong to the
classes IgM, IgG and IgM, respectively.
Furthermore, proteins to which the monoclonal antibodies
produced by OK-l, OK-2 and OK-3 respectively recognize and
bind are as follows.
- Monoclonal antibody produced by OK-1
This antibody recognizes four bands, 10, 68.9 and 86.6
kilodaltones in Western blotting of human Leukemia T cell
(TALL-1) protein.
- Monoclonal antibody produced by OK-2
This antibody recognizes bands of 40 and 90 kilodaltons
in Western blotting of TALL-1 protein.
- Monoclonal antibody produced by OK-3
This antibody recognizes four bands of 86.4, 74.2, 61.4
and 36.4 kilodaltons in Western blotting of TALL-1 protein,
and recognizes three bands of 89, 82 and 45 kilodaltons in
Western blotting of 2FUr protein. *Note, TALL-1 cell line:
see MAMMALIAN CELL CULTURE TECHNOLOGY, edited by M. Shikita
et al. SOFT SCIENCE PUBLICATIONS, Tokyo, pp. 141-162, 1985.
Example 2
Diaqnosis examle by detecting the protein accordinq to
immunofluorescence
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Fibroblasts of a normal human being and a patient of
Down's disease were placed together with a
cover glass (12 x 12 mm) for a microscope in a Petri dish
having a diameter of 35 mm, and cultured. As the medium
5 was used a ~ixture wherein 10% nonessential amino acid
(NEAA), 10 mM sodium pyruvate, 0.1% lactoalbumin
hydrolyzate (LAH) and 10% FCS were contained in MEM. The
cells were cultured at room temperature for
5 days, fixed in a 3.7% formaldehyde solution in PBS
immediately after washing the cover glass 2 to 3 times
with PBS, washed with distilled water, and further fixed
in acetone at -20C.
15 ~Q of the monoclonal antibody obtained in Example l
(antibody derived from hybridoma OK-l, OK-2 or OK-3) was
placed on the fixed cells, and the mixture was allowed to
react at room temperature for one hour. After washing
the mixture three times with PBS, lS ~ of antimouse IgG
antibody or an antimouse IgM antibody, as a second
antibody, labelled with FITC was placed
thereon, the mixture was allowed to react at room
temperature for one hour. The mixture was washed three
times with PBS, once washed with distilled water in order
to remove salts, placed on a slide glass at a state of
the surface fixing the cells down, and sealed with
*Gelvatol to obtain a test sample piece.
The test sample was tested about presence of luminescence
on the surface layer using a fluorescence microscope.
The resulting results are shown in Table 1. Fibroblasts
of the patient of Down's disease emitted fluorescence,
whereas those of the normal human being did not emit.
Thus, it will be appreciated that presence of chromosomal
aberration can readily be diagnosed in a high sensitivity
without using a special technique according to the
present invention.
*Trade ~ark
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In order to confirm the existence of the protein on the
membrane, detection by membrane immunofluorescence
wherein the cells are not fixed and reacted with a
fluorescent dye was also carried out as paralleled to the
above method, and the resulting results were the same as
those obtained above.
Table 1 Presence or absence of luminescence by
immunofluorescence
Monoclonal Fibroblast
antibody Normal human Patient of
bein Down's disease
g
Produced by OK-l - +
Produced by OK-2 - +
Produced by OK-3 - +
Note +: Luminescence was observed
-: Luminescence was not observed
