CA2054302A1

CA2054302A1 - Monoclonal antibody-containing agent

Info

Publication number: CA2054302A1
Application number: CA 2054302
Authority: CA
Inventors: Kazukiyo Onodera; Ryoji Yao; Shinichi Ito
Original assignee: Individual
Current assignee: Shin Etsu Bio Inc
Priority date: 1990-10-29
Filing date: 1991-10-28
Publication date: 1992-04-30
Also published as: JPH05199896A

Abstract

Abstract of the Disclosure The agent of the present invention comprising a monoclonal antibody which is specifically linked to a protein encoded by human chromosome 21 and which is linked to a protein whose expression in brain varies dependent upon age or a protein which is expressed in only specific cells of brain can be obtained as follows.
Mice are immunized with CHO cells containing long arms of human chromosome 21 (2Fur cells), then the resulting antibody-producing cells are fused with mouse myeloma cells and hybridomas which produce monoclonal antibodies capable of specifically recognizing a protein encoded by human chromosome 21 are selected. A hybridoma which produces a monoclonal antibody capable of recognizing a protein expressed in the human brain tissues is selected from the resulting hybridomas and then the hybridoma is proliferated to give the intended monoclonal antibody.

Description

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 :
"

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)~, ' 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 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.

,. ' ' ~;
~' :

Claims

1. An agent comprising a monoclonal antibody which is specifically linked to a protein encoded by human chromosome 21 and which is linked to large pyramidal cells of human brain tissues.

2. The agent of claim 1 wherein the monoclonal antibody is linked to a cell nucleus protein having a molecular weight of 32 kilodaltons whose expression in brain varies dependent upon age.

3. The agent of claim 1 wherein the monoclonal antibody is prepared by immunizing mice with 2Fur cells, that is CHO cells containing long arms of human chromosome 21, fusing the resulting antibody-producing cells with mouse myeloma cells, selecting hybridomas which produce monoclonal antibodies capable of specifically recognizing a protein encoded by human chromosome 21, selecting, from these hybridomas, a hybridoma which produces a monoclonal antibody capable of recognizing a protein expressed in the human brain tissues and proliferating the hybridoma.