JP2001233884A5 - - Google Patents
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- JP2001233884A5 JP2001233884A5 JP2000049448A JP2000049448A JP2001233884A5 JP 2001233884 A5 JP2001233884 A5 JP 2001233884A5 JP 2000049448 A JP2000049448 A JP 2000049448A JP 2000049448 A JP2000049448 A JP 2000049448A JP 2001233884 A5 JP2001233884 A5 JP 2001233884A5
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- 150000001875 compounds Chemical class 0.000 description 23
- 239000000427 antigen Substances 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 229910052760 oxygen Inorganic materials 0.000 description 13
- 229910052717 sulfur Inorganic materials 0.000 description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 239000012634 fragment Substances 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 125000004432 carbon atom Chemical group C* 0.000 description 6
- DOFZAZXDOSGAJZ-UHFFFAOYSA-N disulfoton Chemical compound CCOP(=S)(OCC)SCCSCC DOFZAZXDOSGAJZ-UHFFFAOYSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 230000000984 immunochemical effect Effects 0.000 description 4
- 238000000691 measurement method Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 125000002947 alkylene group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- -1 p-methoxybenzyl group Chemical group 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 1
- 125000002774 3,4-dimethoxybenzyl group Chemical group [H]C1=C([H])C(=C([H])C(OC([H])([H])[H])=C1OC([H])([H])[H])C([H])([H])* 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- MYBBMMORFOKMMQ-UHFFFAOYSA-N acetonitrile;hexane Chemical compound CC#N.CCCCCC MYBBMMORFOKMMQ-UHFFFAOYSA-N 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012230 colorless oil Substances 0.000 description 1
- LGTLXDJOAJDFLR-UHFFFAOYSA-N diethyl chlorophosphate Chemical compound CCOP(Cl)(=O)OCC LGTLXDJOAJDFLR-UHFFFAOYSA-N 0.000 description 1
- SBZXBUIDTXKZTM-UHFFFAOYSA-N diglyme Chemical compound COCCOCCOC SBZXBUIDTXKZTM-UHFFFAOYSA-N 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000005745 ethoxymethyl group Chemical group [H]C([H])([H])C([H])([H])OC([H])([H])* 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- GNOIPBMMFNIUFM-UHFFFAOYSA-N hexamethylphosphoric triamide Chemical compound CN(C)P(=O)(N(C)C)N(C)C GNOIPBMMFNIUFM-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000037 tert-butyldiphenylsilyl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1[Si]([H])([*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 125000006000 trichloroethyl group Chemical group 0.000 description 1
- 125000000025 triisopropylsilyl group Chemical group C(C)(C)[Si](C(C)C)(C(C)C)* 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Description
【特許請求の範囲】
【請求項１】
以下の式（１）：
【化１】
Ａ１は、Ｓ又はＯであり；
Ａ２は、Ｓ、Ｏ又はＮＨであり；
Ａ３は、Ｓ、Ｓ＝Ｏ又はＯ＝Ｓ＝Ｏであり；
Ｒ１及びＲ２は、各々独立に選択されてもよい、枝分かれしていてもよい炭素数１ないし４のアルキル基であり；
Ｒ３は、枝分かれしていてもよい炭素数１ないし４のアルキレン基であり；そして
ｎは１−１０の整数である。］
あるいは、以下の式（２）：
【化２】
Ａ１は、Ｓ又はＯであり；
Ａ２は、Ｓ、Ｏ又はＮＨであり；
Ａ３は、Ｓ、Ｓ＝Ｏ又はＯ＝Ｓ＝Ｏであり；
Ａ４は、Ｏ又はＮＨであり；
Ｒ１及びＲ４は、各々独立に選択されてもよい、枝分かれしていてもよい炭素数１ないし４のアルキル基であり；
Ｒ３は、枝分かれしていてもよい炭素数１ないし４のアルキレン基であり；そして
ｍは１−１０の整数である。］
で表される構造を有する化合物。
【請求項２】
式（１）中、Ａ１及びＡ３がＳであり、Ａ２がＮＨであり、Ｒ１及びＲ２がエチル基であり、そしてＲ３がエチレン基である請求項１に記載の化合物。
【請求項３】
式（２）中、Ａ１及びＡ３がＳであり、Ａ２及びＡ４がＮＨであり、Ｒ１及びＲ４がエチル基であり、そしてＲ３がエチレン基である請求項１に記載の化合物。
【請求項４】
式（１）中、ｎが５である請求項１又は２に記載の化合物。
【請求項５】
式（２）中、ｍが３である請求項１又は３に記載の化合物。
【請求項６】
請求項１ないし５に記載された化合物と高分子化合物又は標識物質との結合体。
【請求項７】
請求項１ないし５に記載の化合物と高分子化合物を結合させることにより抗原を作製し、当該抗原を用いることにより、以下の式（３）：
【化３】
Ａ１は、Ｓ又はＯであり；
Ａ２は、Ｓ、Ｏ又はＮＨであり；
Ａ３は、Ｓ、Ｓ＝Ｏ又はＯ＝Ｓ＝Ｏであり；
Ｒ１、Ｒ２及びＲ４は、各々独立に選択されてもよい、枝分かれしていてもよい炭素数１ないし４のアルキル基であり；そして
Ｒ３は、枝分かれしていてもよい炭素数１ないし４のアルキレン基である。］
で表される構造を有する化合物に反応性を示す抗体を製造することを特徴とする、式（３）で表される化合物に反応性を示す抗体又は抗原と結合可能なそのフラグメントの製造方法。
【請求項８】
請求項６に記載の結合体を抗原として用いることにより製造された、式（３）で表される化合物に反応性を示す抗体又は抗原と結合可能なそのフラグメント。
【請求項９】
モノクローナル抗体である、請求項８に記載の抗体又は抗原と結合可能なそのフラグメント。
【請求項１０】
寄託番号ＦＥＲＭ Ｐ−１７７３２で寄託されているハイブリドーマから産生されるモノクローナル抗体４４Ｋ７−１−１である、請求項８若しくは９に記載の抗体又は抗原と結合可能なそのフラグメント。
【請求項１１】
請求項８ないし１０のいずれか１項に記載の抗体を産生するハイブリドーマ。
【請求項１２】
寄託番号ＦＥＲＭ Ｐ−１７７３２で寄託されている、請求項１１に記載のハイブリドーマ。
【請求項１３】
請求項８ないし１０のいずれか１項に記載の抗体又は抗原と結合可能なそのフラグメントを用いることを特徴とする、式（３）で表される化合物の免疫化学的測定方法。
【請求項１４】
さらに請求項１ないし５のいずれか１項に記載の化合物、又は請求項６に記載の結合体を用いることを含む、請求項１３に記載の免疫化学的測定方法。
[Claims]
(1)
Equation (1) below:
Embedded image
A 1 is S or O;
A 2 is S, O or NH;
A 3 is S, S = O or O = S = O;
R 1 and R 2 are an optionally branched alkyl group having 1 to 4 carbon atoms, which may be independently selected;
R 3 is an optionally branched alkylene group having 1 to 4 carbon atoms; and n is an integer of 1-10. ]
Alternatively, the following equation (2):
Embedded image
A 1 is S or O;
A 2 is S, O or NH;
A 3 is S, S = O or O = S = O;
A 4 is O or NH;
R 1 and R 4 are an optionally branched alkyl group having 1 to 4 carbon atoms, which may be independently selected;
R 3 is an optionally branched alkylene group having 1 to 4 carbon atoms; and m is an integer of 1-10. ]
A compound having a structure represented by the formula:
(2)
The compound according to claim 1, wherein in formula (1), A 1 and A 3 are S, A 2 is NH, R 1 and R 2 are an ethyl group, and R 3 is an ethylene group.
(3)
The formula (2) according to claim 1 , wherein A 1 and A 3 are S, A 2 and A 4 are NH, R 1 and R 4 are an ethyl group, and R 3 is an ethylene group. Compound.
(4)
The compound according to claim 1, wherein n is 5 in the formula (1).
(5)
The compound according to claim 1, wherein m is 3 in the formula (2).
6.
A conjugate of the compound according to claim 1 and a polymer compound or a labeling substance.
7.
An antigen is prepared by binding the compound according to any one of claims 1 to 5 with a polymer compound, and the antigen is used to obtain the following formula (3):
Embedded image
A 1 is S or O;
A 2 is S, O or NH;
A 3 is S, S = O or O = S = O;
R 1 , R 2 and R 4 are independently selected alkyl groups having 1 to 4 carbon atoms which may be independently branched; and R 3 has 1 carbon atom which may be branched. To 4 alkylene groups. ]
A method for producing an antibody reactive with the compound represented by the formula (3) or a fragment thereof capable of binding to an antigen, the method comprising producing an antibody reactive with the compound having the structure represented by the formula:
Claim 8.
An antibody or a fragment thereof capable of binding to an antigen, which is reactive with the compound represented by the formula (3), produced by using the conjugate according to claim 6 as an antigen .
9.
9. The antibody or fragment thereof capable of binding to the antibody according to claim 8, which is a monoclonal antibody.
10.
10. The antibody or fragment thereof capable of binding to an antigen according to claim 8 or 9, which is a monoclonal antibody 44K7-1-1 produced from a hybridoma deposited under accession number FERM P-17732.
11.
A hybridoma that produces the antibody according to any one of claims 8 to 10.
12.
The hybridoma according to claim 11, deposited under accession number FERM P-17732.
Claim 13
An immunochemical measurement method for the compound represented by the formula (3), comprising using the antibody or the fragment thereof capable of binding to the antigen according to any one of claims 8 to 10.
14.
The immunochemical assay method according to claim 13, further comprising using the compound according to any one of claims 1 to 5 or the conjugate according to claim 6.
【０００１】
【発明の属する技術分野】
本発明は、Ｏ，Ｏ−ジエチル−Ｓ−２−（エチルチオ）エチルホスホロジチオエート（一般には、「エチルチオメトン」又は「ジスルホトン」と呼称される：以下、本明細書中「エチルチオメトン」と言う）及びその類縁化合物のハプテン化合物、抗原、抗体及び抗原と結合可能なそのフラグメントに関する。
[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to O, O-diethyl-S-2- (ethylthio) ethyl phosphorodithioate (generally referred to as "ethylthiomethone" or "disulfoton"; hereinafter, referred to as "ethylthiomethone"). And hapten compounds and antigens thereof, antigens, antibodies and fragments thereof capable of binding to the antigen .
本発明はさらに、前記抗原、抗体及びその抗原と結合可能なそのフラグメントを用いた免疫化学的測定方法に関する。 The present invention further relates to an immunochemical measurement method using the antigen, the antibody, and a fragment thereof capable of binding to the antigen .
従来、例えば農作物中のエチルチオメトンは米、果実、野菜、いも類、豆類等から抽出し、精製した後、ガスクロマトグラフィー（ＧＣ）により分析されてきた。即ち、例えば、試料をアセトンで抽出し、ジクロロメタンに転溶し、ヘキサン−アセトニトリル分配してアセトニトリル層を濃縮乾固し、そしてジクロロメタン、アセトンに転溶後、ＧＣで測定する方法等が採用されている（「最新 農薬の残留分析法」 第２３４頁−第２３５頁、中央法規出版 １９９５年４月１日発行））。これらの方法は、試料の調製が煩雑で多大の手順と時間を必要とし、分析に熟練を要すること、並びに、測定装置や設備等に高額の費用を必要とする等の問題点がある。エチルチオメトンの測定は短時間で膨大な数の試料の分析結果を出す必要があり、精度面だけでなく、簡便性、迅速性及び経済性をも具備した新規測定方法が要求されてきている。 Conventionally, for example, ethylthiometon in agricultural products has been extracted from rice, fruits, vegetables, potatoes, beans, etc., purified, and then analyzed by gas chromatography (GC). That is, for example, a method of extracting a sample with acetone, dissolving in dichloromethane, distributing in hexane-acetonitrile, concentrating the acetonitrile layer to dryness, dissolving in dichloromethane and acetone, and measuring by GC, etc. has been adopted. (“Recent Analytical Methods for Pesticide Residues”, pp. 234-235, published by Chuo Hokki Publishing on April 1, 1995). These methods have problems in that the preparation of the sample is complicated, requires a large amount of procedures and time, requires a high level of skill in the analysis, and requires high costs for measuring devices and equipment. In the measurement of ethylthiomethone, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and a new measurement method having not only the accuracy but also the simplicity, the quickness, and the economy is demanded.
本発明は、さらに、エチルチオメトンハプテンと高分子化合物との結合体を提供することを目的とする。
本発明は、さらにまた、前記抗体を産生するハイブリドーマを提供することを目的とする。
Another object of the present invention is to provide a conjugate of an ethylthiomethone hapten and a polymer compound.
Another object of the present invention is to provide a hybridoma that produces the antibody .
本発明は、さらに、前記抗体若しくは抗原と結合可能なそのフラグメント及び／又は前記エチルチオメトンハプテンと高分子化合物若しくは標識物質との結合体を使用することを含む、エチルチオメトンの免疫化学的測定方法を提供することを目的とする。 The present invention further provides an immunochemical measurement method for ethyl thiomethone, which comprises using a conjugate of the antibody or antigen-binding fragment and / or the ethyl thiometon hapten with a polymer compound or a labeling substance. The purpose is to:
反応は、マイナス１０℃から溶媒の沸点、好ましくは２０℃から８０℃で、３０分から１０時間、好ましくは３０分から２時間行う。
Ｐで表されるカルボキシル基の保護基は公知のものでよく、具体例として、例えばメチル基、エチル基、ｔｅｒｔ−ブチル基、ベンジル基、ｐ−メトキシベンジル基、３，４−ジメトキシベンジル基、トリクロロエチル基、トリメチルシリル基、ｔｅｒｔ−ブチルジメチルシリル基、ｔｅｒｔ−ブチルジフェニルシリル基、トリエチルシリル基、トリイソプロピルシリル基、トリメチルシリルエトキシメチル基等が挙げられる。
The reaction is carried out at a temperature of -10 ° C to the boiling point of the solvent, preferably 20 ° C to 80 ° C, for 30 minutes to 10 hours, preferably 30 minutes to 2 hours.
The protecting group for the carboxyl group represented by P may be a known group, and specific examples include, for example, a methyl group, an ethyl group, a tert-butyl group, a benzyl group, a p-methoxybenzyl group, a 3,4-dimethoxybenzyl group, trichloroethyl group, a trimethylsilyl group, tert- butyldimethylsilyl group, tert- butyldiphenylsilyl group, triethylsilyl group, triisopropylsilyl group, trimethyl silyl ethoxy methyl group, and the like.
式（Ｘ５）の化合物の合成のための有機溶媒としては、例えば、アセトニトリル、アセトン、ヘキサン、ペンタン、ベンゼン、トルエン、ジクロロメタン、クロロホルム、１，２−ジクロロエタン、酢酸エチル、ジグリム、Ｎ，Ｎ−ジメチルホルムアミド、ヘキサメチルリン酸トリアミド等、又はこれらの混合溶媒を用いることができる。塩基としては、例えば、前述した式（Ｘ３）の化合物の合成に使用可能なものと同様のものを使用できる。 Examples of the organic solvent for synthesizing the compound of the formula (X5) include acetonitrile, acetone, hexane, pentane, benzene, toluene, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, diglyme, N, N-dimethyl. Formamide, hexamethylphosphoric triamide and the like, or a mixed solvent thereof can be used. As the base, for example, the same bases as those usable for the synthesis of the compound of the above formula ( X3 ) can be used.
エチルチオメトンハプテンと高分子化合物との結合は、例えば、活性化エステル法（Ａ．Ｅ．ＫＡＲＵ ｅｔ ａｌ．：Ｊ．Ａｇｒｉｃ．Ｆｏｏｄ Ｃｈｅｍ．，４２，３０１−３０９（１９９４））、又は混合酸無水物法（Ｂ．Ｆ．Ｅｒｌａｎｇｅｒ ｅｔ ａｌ．：Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２３４，１０９０‐１０９４（１９５４））等の公知の方法によって行うことができる。 The bond between the ethyl thiometon hapten and the polymer compound can be determined by, for example, the activated ester method (AEKARU et al . : J. Agric. Food Chem. , 42, 301-309 (1994)) or a mixed acid anhydride. (BF Erlanger et al .: J. Biol . Chem ., 234, 1090-1094 (1954)) and the like.
（ｄ）の工程に用いることのできるミエローマ細胞としては、例えば、Ｂａｌｂ／ｃマウス由来骨髄腫細胞株のＰ３／Ｘ６３−Ａｇ８（Ｘ６３）（Ｎａｔｕｒｅ，２５６，４９５−４９７（１９７５））、Ｐ３／Ｘ６３−Ａｇ８．Ｕ１（Ｐ３Ｕ１）（Ｃｕｒｒｅｎｔ Ｔｏｐｉｃｓ ｉｎ Ｍｉｃｒｏｂｉｏｌｏｇｙ ａｎｄ Ｉｍｍｕｎｏｌｏｇｙ，８１， １−７（１９８７））、Ｐ３／ＮＳＩ−１−Ａｇ ４−１（ＮＳ−１）（Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．，６，５１１−５１９（１９７６））、Ｓｐ２／０−Ａｇ１４（Ｓｐ２／０）（Ｎａｔｕｒｅ， ２７６，２６９−２７０（１９７８））、ＦＯ（Ｊ．Ｉｍｍｕｎｏ．Ｍｅｔｈ．，３５， １−２１（１９８０））、ＭＰＣ−１１、Ｘ６３．６５３、Ｓ１９４等の骨髄腫株化細胞、あるいはラット由来の２１０．ＲＣＹ３．Ａｇ １．２．３．（Ｙ３）（Ｎａｔｕｒｅ， ２７７，１３１−１３３，（１９７９））等を使用できる。 Examples of myeloma cells that can be used in the step (d) include P3 / X63-Ag8 (X63) of Balb / c mouse-derived myeloma cell line (Nature, 256, 495-497 (1975)) and P3 / X63-Ag8. U1 (P3U1) (Current Topics in Microbiology and Immunology, 81, 1-7 (1987)), P3 / NSI-1-Ag 4-1 (NS-1) (Eur. J. Immunol., 6, 511-519). (1976)), Sp2 / 0-Ag14 (Sp2 / 0) (Nature, 276, 269-270 (1978)), FO (J. Immuno. Meth., 35, 1-21 (1980)), MPC-11 , X63.653, S194, etc., or rat-derived myeloma cell lines. RCY3. Ag 1.2.3. (Y3) (Nature, 277, 131-133, (1979)) and the like can be used.
化合物（３）の合成
３．８ｇ（１５ｍｍｏｌ）の化合物（２）とクロロリン酸ジエチル３．０ｇ（１５ｍｍｏｌ）をアセトニトリル１５０ｍｌに溶解して、炭酸カリウム２．１ｇ（１５ｍｍｏｌ）を加えて５０℃で１時間撹拌した。反応混合物をセライトろ過、濃縮してシリカゲルカラム（ｎ−ヘキサン：酢酸エチル＝４９：１→１９：１→９：１）で精製し、無色油状物として２．２ｇ（収率３６％）の化合物（３）を得た。
Synthesis of compound (3) 3.8 g (15 mmol) of compound (2) and 3.0 g (15 mmol) of diethyl chlorophosphate were dissolved in 150 ml of acetonitrile, and 2.1 g (15 mmol) of potassium carbonate was added. Stirred for hours. The reaction mixture was filtered through celite, concentrated and purified by a silica gel column (n-hexane: ethyl acetate = 49: 1 → 19: 1 → 9: 1) to give 2.2 g (yield 36%) of the compound as a colorless oil. (3) was obtained.
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