Note: Descriptions are shown in the official language in which they were submitted.
MONOCLONAL ANTIBODY -CONTAINING AGENT
Background of the Invention
The present invention relates to an agent which
comprises a monoclonal antibody which can recognize a
protein encoded by chromosome 21 and expressed in the human
brain, which i~ linked thereto and which is effective for
evaluating the function of the human brain and the degree
of aging thereof.
Recently, cranial nerve system disorders
represented by aphronesia is becoming a socially and
medically important problem as an abrupt increase of the
population in the aged persons. For this reason, there has
been desired to elucidate the changes of brain functions
associated with the aging phenomenon. ~owever, the ~rain
is one of the greatest ~lack boxes in the living body.
TAe human brain has~been ~tudied in detail while maki~g
use o~ anatomical and histological means. As a re~ult, the
morphological knowledges about the brain have almost been
elucidated. There has presently b~en focused on the
elucidation of the correlation between these morphological
knowledges and ~unctions of the ~raln.
One of the methods for elucidat1ng the
correlation between the morphology and functions of the
brain is a histochemical or cytochemical approach. This
,.
~ ' ` ,
~0~3~
approach is a method for detecting, in situ, a specific
function or propert~ of the h~ain and the approach can
make clear a specific function or property thereof through
direct microscopic obser~ation thereof. More speci~ically,
sites of the brain e~hibiting a specific function or
property are selectively stained so as to differentiate
it from others.
There have been used monoclonal antibodies in
recent histochemical studies o the brain. The monoclonal
antibody is a single protein which can recognize only the
specific substance (antigen) and can specifically be
linked to the substance. The monoclonal antibody is
considered to be an important tool for s~udying brain
functions because of this property. In fact, knowledges
of brain functions have rapidly been increased due to the
histochemical studias which make use of monoclonal
antibodies. For instance, only the cholinergic nerve which
synthesizes acetylcholi~e can selectively be stained by
reacting a monoclonal antibody to choline acetyltransferase
serving as a primary antibody with a section of the brain
tissue, then combining it with an enzyme~labelled secondary
antibody and subsequently reacting ~he product with the
substrate for the enzyme.
Thus, monoclQnal antibodies can widely be used
~or detecting cranial nerve system disorders. It has
become c}ear that chro~osome 21 is the causal ~ene for
2~3~3
Alzheimer's disease which has become of interest la~ely.
In ad~itian, the Down's ~yn~ome is a dlsease in whlch the
nerve cells grow a~no~mally and the aging of the brain
is accelerated. This Down's syndrome is also caused due to
a genetic abnormality calle~ trisomy wherein chromosome 21
is three chromosomes. This probably indicates that some
of proteins encoded by genes present on chromosome 21
would be involved ln functions and growth of nerve systems.
For this reason, there have long been required for the
development of agents which can recognize proteins
encod~d by this chromosome 21 and changes in the expression
of brain protein due to aging and aphranesia.
Summary of the Invention
Accordingly, an ob~ect of the pre~ent invention
is to provide an agent comprising a monoclonal antibody
which can recognize proteins which are coded ~y human
chro ~ome 21 and more particularly can ~ecognize a protein
whose expre~ion in the human brain varies depending on
the age or a protein which is e~pre~ed only in a ~pecific
cell o~ the brain.
The age~t which permits the achievement of the
~oregoing o~ect acco~ding to the .pre~ent invention
co~pr~se~ a ~onoclonal antibody which is ~pecifically
linked with a protein encoded by the human chromosome 21
'
~43~2
and large pyramidal cells of the human brain tissue. The
monoclonal antibody included in the agent according to
the present $nvention c~n specifically liDk~d with the
cell nucleus protein which has a molecular weight of 32
kilodalton (KDa) and whose quantity of expression in the
human brain varies depending on the age.
The monoclonal antibody-containing agent of the
pre~ent invention can be used as an important diagnostic
agent for histochmical studies which is effective in
detecting cranial nerve system disorders due to
abnormalitles in chromosoma 21.
Further, the amino acid sequence of the protein
which is recognized by the monoclonal antlbody included
in the a~e~t can be used in the screen~ng of cDNA library
(a series of complementary DNA's obtaine~ ~y reverse
transcription of mRNA) and is effective for the
elucidation of genes which are expressed and function in
the brain.
Brief Explanation of the Drawings
Fig. 1 ~ ~ a diagram for showing the confirmation
of the presence of a protein which can be linked with
YO-1 in immu~nblot analysî~;
F~g ~ is a diagram shnwing sites in a cell a~
which a ~pecific protein is localized, the protei~ be~n~
'
20~3~2
linked with YO-l in immunoblot analysis;
Fig. 3 is a diagram for showing the confirmation
of the presence of a protein which can be linked with
YO-3 in immunoblot analysis; and
Fig. 4 is a dia~ram for confirming the fact
that YO-l and YO-3 can recognize the YO-l-linked protein
in immunoblot analysis.
In these figures, each reference numeral is as
follows: reference numeral 1 represents the analysis of
2Fur cell with YO-l; Z the analysis of C~10 cell with
YQ-l; 3 the analysis of a second cell nucleus e~tract of
the 2Fur cell with YO-l; 4 the analysis of a first cell
nucleus e~tract of the 2Fur cell with YO-1; 5 the analysis
of S-100 of the 2Fur cell with YO-l; 6 the analysis of
the 2Fur cell with YO-3; 7 the analysis of the CHO cell
with YO-3 8 the analysi~ of an YO-l-linked protein with
YO-l; and 9 the analysis of the YO-l-linked protein with
YO-3.
Detailed E~planation of the Invention
The monoclonal antibody included in the agent
o~ the present invention can be prepared in the following
manner.
Mice are immunized with 2Fur cells (CHO cell~ -
containing long arms of the human chromosome 21) to give
. '" ' ' , . ,
2 ~ 2
antibody-producing cells, the antibody-producing cells are
fused with mouse myeloma cells and then hybridomas which can
produce the monoclonal anti~odies capable of specifically
recognizing a protein encoded by the human chromosome 21.
A hybridoma which can produce a monoclonal antibody capable
of recognizing a protein e~pressed in the cells of the
human brain is then isolated from the resulting hy~ridomas.
The hybridoma can be proliferated to give the foregoing
monoclonal antibody.
Example~ of the present invention will
hereinafter ~e described but the present invention should
not be limited by these e~amples.
E~ample 1
Preparation o~ Monoclonal Anti~od~ YO-l
2Fur cells (107 cells; CHO cells including long
arms of the human chromosome 21; D. Patterson et al., Som.
Cell Genet., 1~75, 1, p.91) were intraperitoneally injected
into BALB/c mice every two weeks over three time~. One
week after the injection, the mice were subjected to booster
in the same manner. Three d~ys after the booster, the
spleens were removed from the mice, suspendea in a DF medium
(containing, in 1,000 ml of the medium, 5.25 g nf Dulbecco-
Modified EAgle ~DME) medium, 5.57 g of Hum F12 ~F12), 3.57 gof HEPES (2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acia), o. 1 g of Streptomyein, 10~ units of Penicillin and
2~3~2
1.4 g of NaHC03), mixed with myeloma cells (P3~X63-Ag8.Ul)
in a ratio of 10:1 and centrifuged at 1,600 rpm for
5 minutes. After adding 50% polyethylene glycol and gently
stirring the resulting mixture, the cells were washed
through centrifugation (1,000 rpm, 10 min). Thereafter,
the spleen cells and the myeloma cells (10b cells~ml) were
suspended in a DF medium containing 15% fetal calf serum,
0.1 ml thereof was dispensed in a plate provided with 96
wells and cultured in a CO~-cultivation apparatus for one
day. Therea~ter, these cells were cultured in a HAT
medium (a medium obtained by dissolving 1.36 mg of
hypoxanthine, 0.39 mg of thymidine and 0.0182 mg of
aminopterin in 100 ml of the DF medium).
The selection of hybridomas was carried out by
dissolving 2Fur cells of C~O cells in a lysing buffer
(a solution containing 0.5844 g of NaCl, 0.4 ml of NP40
(NONIDET P-40; available from Sigma Company) and 0.5 ml
o~ aprotinin in 100 ml of a 10 mM Tris-HC1 buffer (pH 8.0~
absorbing the result-ing solution onto a plate provided
with 96 wells, sub~ecting the culture supernatant to
enz~me-linked immunosorbent assay (ELISA) with respect to
the resulting plate and finally selecting a 2Fur-positive
and CHO-negative clone.
The resulting clone was prolifsrated in the
peritoneal cavity of BALB/c mice and the resulting ascites
~ere used as a crude monoclonal antibody solution. To
.; ., . , . ~
c~nfirm whether the monoclonal antibody thus ohtained
could ~pecifically recogni~e the protein encoded by the
human chromosome 21, the 2Fur cells and the CHO cells
were sub~e~ted to immunoblotting ln the following manner.
The 2Fur cells or the CHO cells were di~solved in the
same lysing buffer and then the re~ulting solution wa~
subjected to SDS ~sodium dodecyl sulfate3-polyacrylamide
gel electrophore~is according to the method of Laemmli
(see Nature, 1970, 227, p. 6B0). After the electroph~re~is,
the SDS waæ removed from the ~el a~d th~ protein was
transferred to a nitrocellulose film by applying an
electric current (a constant current of 150 mA) for 2
hours using a transfer apparatus (aYaila~le from Toyo
Company). After the tran~fer, blocXing was carried out
using a 5% ~kimmil~ solution. The nitrocellulose film was
waæhed with T~S (50 mM Tri~-HCl, 200mM NaCl, pH 7.4
containing 0.05% Tween 20 and then reacted in the crude
m~noolonal antibody solution ~iluted 1,000 time~ with TBS
at 37 C for 2 hours. After washing, the reaction product
was reacted with alkaliphosphataselabelled anti-mou~e IgG
(Tago) at 37 C for 2 hours. Nitroblue te$aazoliu~ and
5-br~mo-4-chloro-3-i~dolyl phosp~ate were used for the
color~tion.
Result~
As ~hown in Fig. 1, there was obtained ~o~oclon~l
2~3~
antibody YO-l (FERM ~P-3123; hereinafter referred to as
~YO-l~) capable of specifically recogniz~ng the protein
having a molecular weight of 32 KDa derived from the 2Fur
cell. Thi~ protein is not expressed in the CHO cell. Thi~
fact clearly indicates that the foregoing protein is
encoded by a gene present on the human chromosome 21.
E~pression of Y0-1-linked Protein in Brain
In the preparation of specimens of brain
ti~sues, a frozen section wa~ prepared by cutting frozen
~rain tissue into pieces having a thickness ranging from
10 to 20 ~m with a cryostat and then adhered to a slide
glass on which egg albumin had been coated and, on the
other hand, the slide glass on which paraffin is mounted
was im~er~ed twice in xylene for 1~ minutes for the
removal of the paraffin and then treated with ethanol and
distilled water for the removal of the xylene.
A~ter reacting the section of ~he brain tissue
adhered to the slide glass with YO-1, the tissue wa~
stained according to the avidinbiotin complex Method
(hBC method) or peroxidase-antlperoxidase method ~PAP
method). Then the section was ~ub~ected to the counter
staining with hematoxylin, treated with ethanol and
~ylene and then sealed with canada bal~am. The~e specimens
were examined by a liyht microscope.
Results
The YO-1-linked protein was e~pressed in large
pyramidal neuron of nerve cells in the brain tissue.
There was not observed any expression in the granulocytes.
etection of Proteins Whose Expression is Dependent Upon Age
In the same manner used in the foregoing
expression in the brain, brain tissues derived from normal
persons and patients suffering from Down's disease, of
various ages, were reacted with YO-l, the tissues were
stained and e~amined through microscopic obæervation.
Results
Table 1 shows the results of YO-l-linked protein
expression in the large pyramidal cells of the brain. The
YO-l-linked protein e~pression was positiYe in all the
e~amined normal persons of 2 years old or older. On the
other hand, it was already expressed in the brain of
foetus of 40-weeks-old in case of patients suffering from
Down 7 S disease. Its e~pression was positive in both
pyramidal cells and granuloc~tes in case of anti-supero~ide
dismutase (SOD) monoclonal an ibody used as a control. In
the latter c~se, there was not observed any difference
between the normal persons and the patient~ suffering from
Down's disea~e.
Moreover, glia cells in the brain was likewise
:.
2 0 ~ 2
examined through ~icroscopic observation. Table 2 shows
the results of the YO-1-linked protein expression in the
glia cells. The YO-1-linked protein expression was negative
in astrocytes, oligodendrocytes and microglia. On the
other hand, if staining was performed with anti-SOD
monoclonal antibody as a control, positive glia ~ells
were detected in both normal persons and patients
suffering from Down ' 5 disease, of 40-week-old (foetus) or
older.
Table 1
YO-l Anti-SOD'
Death A~e Normal Down's Disease Normal Down's Disease
17 weeks foetus - -
25 weeks foetus
40 weeks foetus - + ~ + +
3 months infant - ~ + +
2 years infant + + + + +
32 years adult + + ~ ~ ~ + ~
+ : weakly expressed; + : expressed in a part of cell~;
+ + : e~pressed ; - : not e~pressed.
'Anti-SOD: anti-superoxide dismNtase.
3 ~ ~
Table 2
YO~1 Anti-SOD-
Death Age Normal Down's Disease Normal Down's Disease
17 weeks foetus - -
25 weeks foetus - -
40 weeks foetus - - -~ ~ +
3 months inf ant - - + + ~
2 years infant - - + ~ + +
32 years adult - ~ + + ~
+ : weakly e~pressed; ~ : e~pressed in a part of cells;
+ + : expressed ; - : not e~pressed.
Anti-SOD: anti-superoxide dismutase.
Sites at Which YO-l linked_Protein is Localized
A buffer solution A (10 mM HEPES, p~ 7.9; 1~5 mM
MgCl2; and 10 mN DTT) was added to 2Fur cells in an amount
of 10 ml per lx 10l cells, the resulting mixture was
homogeni~ed 10 times with a Teflon homogenizer and then
centrifuged at 2,000 rpm for 10 minutes to give a
supernatant which was referred to as S-100 fraction. To
the precipitates, there was added 30 ml of a bufer
solution C (20 mM HEPES, pH 7.9; 25% glycerol; 0.42 M NaCl;
1.5 ~M MgCl2; a. 2 mM EDTA; 0.5 mM PMSF; and 0.5 mM DTT),
the resulting mi~ture was homoyenized 10 times with a
Te1On homoge~izer and then centrifu~ed at 16,00Q rpm for
20 minutes to give a ~upernatant which was referred to as
first cell nucleus extract. The precipitates were
2 ~
further suspended in 40 ml o~ the same buffer solution C,
the resulting mixture was homogenized 10 times with a
Teflon homogenizer and then centrifuged at 35,000 rpm
for 30 minutes to give a ~upernatant which was referred
to as a second cell nucleus extract. The S-100 ~raction
and the fir~t and second cell nucleus e~tracts each was
dialyzed against phosphate buffered saline (PBS) at 4 C
overnight. These various protein ~ractions were separat~d
by SDS-polyacrylamide gel electrophoresi~ rs~pectively
and subjected to immunoblotting in which YO-1 was used a~
a primary antibody.
Re3ults
The protein having a molecular weight of 32 KDa
which was rPcognized by YO-l was presen~ in the second
cell ~ucleus e~tract a~ shown in Fig~ 2. Thus, it was
elucidated that YO-l was linked with the cell nucleu~
protein.
Purification of YO-l-Linked Protein
. .
YO-l ~erived from the mouse ascites ~4 mg, 10 ml)
which had been purified by Affî-gel protein A (avail~ble
from Bio-Rad) and dialy~ed against PBS at 4 C for 3 hours
was suspended in O.6 ~ of swollen CNBr-activated Sepharose
4B (available from Pharmasia), incubated at 4 C ov~rni~ht
to ad~orb YO-1 onto the CNBr-activat~d Sepharo~e 4~ and
20~3~
to thus from YO-I-Sepharose 4B.
The resulting YO-l-Sepharose 4B was packed in a
column, washed with PRS and then the second cell nucleus
extract wa~ passed through the column at a flow rate of
lOml/hr to adsorb proteins onto the column. Elution was
performed using 0.1 M glysine-HCl (pH 2.5~. The proteins
eluted were neutralized with 2 M Tris-HCl (p~ 8.0~. The
resulting proteins were further purifled by reverse phase
high performance liquid chromatography (reverse pha~e
HPLC; TSK gel Phenyl-5PW, a~ailable rom Tosoh Corporation).
The proteins were concentrated to dryness by lyophilization,
dissol~ed in 25 mM Tris-HCl (pH 8.S) containing 1 mM EDTA
~ollowed by the addition of an endoproteinasa Lys-C
(available from Behringer Manheim) in an amount of lJ10
time that of the proteins ~nd reaction performed at 37 C
overnight. Peptide ~ragment~ obtained after the digestion
were isolated by TSK gel ODS-120T (available from Tosoh
Corporation). Each peptlde fragment isolated was
concent~ated to dryness by a centrifugal evaporator,
di~solved in ultrapure water a~d then the amino acid
sequence thereof was det~nmined ~y a Gas Phase Prot~in
Sequencer (~h1mazu PQS~
Results
As a result of the reverse pha~e HPLC separation,
it was ~ound that the YO-l-linXed protein having a ~olecular
1 4
:
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2 ~ ~ ~ 3 ~ 2
weight of 32 KDa was decomposed into about 30 peptide
fragments. Peptid~ fragments showing main 8 pea~s had th~
following amino aoid sequences.
No. 12: TPK; No. 21: ATGSATPK;
No. 22: KPAAAAVTK; No. 24: GTGASGSFK;
No. 27: LGLK; No. 29: ALAAAGYDVEK;
No. 30: ERSGVShAALK; No. 31: ASGPPVS~LITK.
Each symbol stands for the following amino acid
residue (Each No. represents the order of the corresponding
peak appeared).
A: Alanine; C: Cysteine;
D: Aspartic acid; E: Glutamic acid;
F: Phenylalanine; G: Glycine;
H: Histidine; I: I~oleucine;
K. Lysine; L: ~eucine;
M: Methionine; N: A~paragine;
P: Proline; Q: Glut~mine;
R: Arginine; S: Serine;
T: Threonine; V: Val ine;
W: Tryptophan; Y: Tyrosine.
E~ample 2
Preparation o-f Monoclonal Antibody YO-3
2Fur cells ( 107 cells; CHO cells including long
arms o~ the human chromosome 21; ~. Patters2n et al., Som.
Cell GenetO, 1975, 1, p. 91) were intraperi~oneally injected
2~3~
into BALB/c mice every two weeks over three times. One
week after the injection, the mice were subjected to ~ooster
in the same manner. Three days after the booster, the
spleens were removed from the mice, suspended in a DF
medium (containing, in 1,000 ml of the medium, 5.25 g of
Dulbecco-Modi~ied Eagle (DME) medium, 5.57 g of Hum F12
(F12), 3.57 g of HEPES (2-hydro~yethylpiperazine-N'-2-
ethanesulfonic acid), 0.lg of Streptomycin, 10~ units of
Penicillin and 1.4g of NaHCO~), mixed with myeloma cells
(P3/X63-Ag8.U1) in a ratio of 10:1 and cenfrifuged at
1,600 rpm for 5 minutes. After adding 50% polyethylene
glycol and gently stirring the resulting miYture, the cells
were washed through centrifugation ~1,000 rpm, 10 min).
Thereafter, the spleen cells and the myeloma cells
(10~ cells/ml) were suspended in a DF medium containing 15%
fetal calf serum, 0.1 ml thereof was dispensed in a plate
provided with 96 wells and cultured in a CO2-cultivation
apparatus for one day. Thereafter, these cells were cultured
in a HAT medium (a medl~m obtained by dis~olving 1.36 mg
of hypoxanthine, 0.39 m~ of thymidine and 0.~182 of
aminopterin in 100 ml of the DF medi~m).
The selection of hybridomas was carried out by
dissolving 2Fur cells or CH~ cells in a lysing buffer (a
solution containing V.5844 g of NaCl, 0.4 ml of NP40
(NONIDET P-40; available from Sigma Company~ and 0.5 ml
of aprotinin in 100 ml o~ a 10 mM Tris-~Cl buffer (p~ 8.0)~,
1 6
'
adsorbing the resulting solution onto a plate proYided
with 96 wells, subjecting the culture supernatant to
enzyme-lin~ed immunosorbent assay (ELISA) with respect to
the resulting plate and finally selecting a 2Fur-positive
and CH0-negative clone.
The resulting clone was proliferated in the
peritoneal cavity of BAL~/c mice and the resulting ascites
were used as a crude monoclonal antibody solution. To
confirm whether the monoclonal antibody thus ohtained
could specifically recognized the protein encoded by the
human chromosome 21, the 2Fur cells and the CHO cells
were subjected to immunoblotting in the following manner.
The 2Fur cells or the CH0 cells were dissolved in the
same lysing buffer and then the resulting solution was
subjected to SDS (sodium dodecyl sulfate)-polyacrylamide
gel electrophoresis according to the method oE Laemmli
(see Nature 1370, 227, p. 680~. After tAe electrophoresis,
the SDS was removed from the gel and the protein was
transferred to a nitrocellulose film by applying an
electric current (a constant current of 150 mA) for 2 hours
using a transfer apparatus ~available from Toyo Company~.
After the transfer, blocking was carried out using a 5%
skimmil~ solution. The nitrocellulose film was washed
with TBS ( 50 mM Tr~s-HCl~ 200 mM NaCl, pR 7.4) containing
O.05~ Tween 20 and then reacted ln the crude monoclonal
antibody solution diluted 1,000 times with TBS at 37 C
2 0 ~ 2
for 2 hours. After washing, the reaction produc~ was
reacted with alkaliphosphataselabelled anti-mouse IgG
(Tago) at 37 C for 2 hours. Nitroblue tetraazolium and
5-bromo-4-chloro-3-indolyl phosphate were used for the
coloration thereof.
Results
As shown in Fig. 3, there was obtai~ed a
monoclonal antibody YO-3 (FERM BP-3423; hereinafter referred
to as ~YO-3") capable of specifically recognizing the
proteîns having molecular weights of 32 KDa and 23 KDa
respectively derived from the 2Fur cell and the protein
having a molecular weight of 23 KDa derlved form the CHO
cells.
Expression of YO-3-linked Protein in Brain
.
Specimens of brain tissue were prepared as
follows. A frozen sect~on was prepared by cutting frozen
brain tissue into pieces havin~ a thickness ranging from
10 to 20 ~m with a cryostat and then adhered to a slide
glass on which had ~een coated and, on the other hand, a
paraffin section mounted on a slide glass was immersed
twice in ~ylene for 10 minutes to remove the paraffin and
then treated with ethanol and distilled wster to remove
the xylene.
After reacting the s~ction of the brain tissue
2 ~
adhered to the slide glass with YO-3, the tissue was
stained according to the avidin-biotin complex method (ABC
method) or peroxidase-antipero~idase method ~PAP method).
Then the section was sub~ected to the counter staining
with hemato~ylin, treated with ethanol and xylene and
then sealed with canada balsam. These specimens were
examined by a light microscope.
Rasults
The YO-3-linked protein was e~pressed in large
pyramldal neuron of ner~e cells in the brain tissue like
the YO-l-linked protein. There was not observed any
e~pre~sion in the small granulocytes.
etection of Proteins Whose E~pression is ~ependent Upon A~e
In the same ma~ner used in the foregoing
expression in the brai~, brain tissue derived from normal
persons and patients sufering from Down's disease, of
various ages were reacted with YO-3, the tissues were
stained examined through microscopic observation.
Re~ults
Table 3 shows the re~ults of YO-3-lin~ed
protein expr~ssion in the large pyramidal cells of the
brain. The YO-3-linked protein e~pression w~s positive in
both eYam~ned normal per~on~ and pakients suffering from
1 g
,
2~30~
Down's disease, of 40-week-old or olde~. T~ere was not
observed any e~pression of the YO-3-linked protein in the
glia cells of the brain as in the case of the YO-l-linked
protein.
Table 3
YO-3 Anti-SOD'
eath Age Normal Down's Disease Normal Down's Disease
17 weeks foetus - - :
25 weeks foetus
40 weeks foetuæ ~ ~ ~ + +
3 months infant +
2 years infant ~ + + + + +
32 years adult + + + + + ~ + ~
~ : weakly expressed; + : expressed in a part of cells;
+ ~ : e~pressed ; - : not expressed.
~Anti-SO~: anti-superoxide dismutase.
Homolo~y of YO-1-Linked Proteln and YO-3-Linked Protein
YO-1 derived from the mouæe a~cites ~4 mg, 10 ml)
which had been purified by Affi-gel protein A (available
from Bio-Rad) and dialyzed against PBS at 4 C for 3 hours
wa~ suspended in 0.6 g of sw~llen CNBr-activated Sepharose
4B ~availabl~ from Pharmasia), incubated at 4 C o~ernight
to adæorb ~0-1 onto the CN~r-activated Sepharose 4B a~d
to thus ~orm YO-1-Sepharose 4B.
The resulting YO-1-Sepharose 4B was packed in a
2 0
3 ~ 2
column, washed with P~S and then the second cell nucleus
e~tract was pas~ed through the column at a flow rate of
10 mlJhr to adfiorb protein~ onto the column Elution was
performed using 0.1 M glysine-HCl (pH 2.5). The protain~
eluted were neutralized with 2 M Tri~-HCl ~pH 8.0). The
resultlng proteins were further purlfied by reverse pha~e
high performance liquid chromatography (reverse phase HPLC;
TSX gel Phenyl-5PW; availa~le from To~oh Corporation).
The purified protein~ were dis~olved in the
ly~ing buffer and then the resulting solut~on was subjected
to SDS l~odium dodecyl sulfate~ polyacrylamide gel
electrophore~is according to the method of Laemmli (see
Nature, 1970, 227, p. 6~0). After the electrophoresis,
the SDS was removed from the gel and the protein was
transferred to a niirocellulosa fil~ by applying an
electric current (a constant current of 150 mA) for ~ hours
using a tran~fer apparatus (available from Toyo Company~.
Arter the transPer, the nitrocellulo~e fiim was ~locked
with a 5~ skimmilk solution~ The nitrocelluios~ îilm wa~
wasned with TB5 ~50 mM Tris-HCl, 200 mM NaCl, p~ 7.4)
containing 0.05~ T~een 20 and the~ reacted in a ~olution
containing YO-3 diluted with TB3 at 37 C for 2 hours.
A~ter wa~hing, the reaction producL was reacted witn
alkalipho~p~ata~elabelled anti-mouse IgG [Tago) at 37 C
for 2 hours. Nitro~lue tetraazoli~m and 5-bromo-4-chloro-
3-indolyl pho~phate were used for tne coloration thereo~.
- ~ ,
~ ~ -
,
~0~3~2
By way of comparison, immunoblotting in which
YO-1 was used as a primary antibody was also carried out.
Results
-
As shown in Fig. 4, YO-3 could recognize the
YO-l-linked protein. Thus, it is confirmed that the
YO-1-linked protein of 32 KDa specific to the 2Fur call
is identical to the YO-3-linked protein.
2 2
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