JPH0830059B2 - Bilirubin derivative - Google Patents
Bilirubin derivativeInfo
- Publication number
- JPH0830059B2 JPH0830059B2 JP10549687A JP10549687A JPH0830059B2 JP H0830059 B2 JPH0830059 B2 JP H0830059B2 JP 10549687 A JP10549687 A JP 10549687A JP 10549687 A JP10549687 A JP 10549687A JP H0830059 B2 JPH0830059 B2 JP H0830059B2
- Authority
- JP
- Japan
- Prior art keywords
- bilirubin
- residue
- formula
- group
- substituent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical class N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 title claims description 101
- 125000001424 substituent group Chemical group 0.000 claims description 28
- 239000002262 Schiff base Substances 0.000 claims description 15
- 150000004753 Schiff bases Chemical class 0.000 claims description 15
- 150000001875 compounds Chemical group 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical group O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 6
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical group N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 6
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical group O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 claims description 5
- 125000002947 alkylene group Chemical group 0.000 claims description 5
- 125000000524 functional group Chemical group 0.000 claims description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Chemical group OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Chemical group OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims description 2
- 125000004434 sulfur atom Chemical group 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims 2
- 238000000034 method Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 10
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 9
- 229960003151 mercaptamine Drugs 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 125000003277 amino group Chemical group 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 6
- 235000018417 cysteine Nutrition 0.000 description 6
- 108060003552 hemocyanin Proteins 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 230000001588 bifunctional effect Effects 0.000 description 4
- -1 dialdehyde compound Chemical class 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000012279 sodium borohydride Substances 0.000 description 4
- 229910000033 sodium borohydride Inorganic materials 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 3
- 241001529572 Chaceon affinis Species 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940097043 glucuronic acid Drugs 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 206010023126 Jaundice Diseases 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000000984 immunochemical effect Effects 0.000 description 2
- 238000007449 liver function test Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- JXSSPZOCHGZWJP-UHFFFAOYSA-N 1,2-difluoro-3,4-dinitrobenzene Chemical class [O-][N+](=O)C1=CC=C(F)C(F)=C1[N+]([O-])=O JXSSPZOCHGZWJP-UHFFFAOYSA-N 0.000 description 1
- VILFTWLXLYIEMV-UHFFFAOYSA-N 1,5-difluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC([N+]([O-])=O)=C(F)C=C1F VILFTWLXLYIEMV-UHFFFAOYSA-N 0.000 description 1
- LBLYYCQCTBFVLH-UHFFFAOYSA-N 2-Methylbenzenesulfonic acid Chemical compound CC1=CC=CC=C1S(O)(=O)=O LBLYYCQCTBFVLH-UHFFFAOYSA-N 0.000 description 1
- BKOOMYPCSUNDGP-UHFFFAOYSA-N 2-methylbut-2-ene Chemical group CC=C(C)C BKOOMYPCSUNDGP-UHFFFAOYSA-N 0.000 description 1
- QLHLYJHNOCILIT-UHFFFAOYSA-N 4-o-(2,5-dioxopyrrolidin-1-yl) 1-o-[2-[4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxobutanoyl]oxyethyl] butanedioate Chemical compound O=C1CCC(=O)N1OC(=O)CCC(=O)OCCOC(=O)CCC(=O)ON1C(=O)CCC1=O QLHLYJHNOCILIT-UHFFFAOYSA-N 0.000 description 1
- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical group OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 1
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical group OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical group [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- PCSMJKASWLYICJ-UHFFFAOYSA-N Succinic aldehyde Chemical compound O=CCCC=O PCSMJKASWLYICJ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 1
- NXVYSVARUKNFNF-UHFFFAOYSA-N bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxybutanedioate Chemical compound O=C1CCC(=O)N1OC(=O)C(O)C(O)C(=O)ON1C(=O)CCC1=O NXVYSVARUKNFNF-UHFFFAOYSA-N 0.000 description 1
- VYLDEYYOISNGST-UHFFFAOYSA-N bissulfosuccinimidyl suberate Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)C(S(O)(=O)=O)CC1=O VYLDEYYOISNGST-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- IAQRGUVFOMOMEM-UHFFFAOYSA-N but-2-ene Chemical group CC=CC IAQRGUVFOMOMEM-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000004678 hydrides Chemical class 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 150000004032 porphyrins Chemical group 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000007723 transport mechanism Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Saccharide Compounds (AREA)
- Pyrrole Compounds (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） この発明は、ビリルビン誘導体に関するものである。TECHNICAL FIELD The present invention relates to a bilirubin derivative.
（従来の技術） 生体内で機能を失ったヘモグロビン等のヘム蛋白のヘ
ムは、その鉄原子を回収し再利用するために、そのポル
フィリン環が開環されてビリベルジンに変換され、次い
で還元されてビリルビンになる。ビリルビンは水に溶け
難く、これを生体外に排泄するには抱合されなければな
らない。現在抱合体としては、グリクロン酸抱合体、グ
ルコース抱合体、キシロース抱合体、抱合体"C"等が知
られている。このビリルビンの抱体または排泄が何らか
の原因で障害を受けた病態は黄疽と呼ばれていて、肝臓
をはじめとする消化器疾患、血液疾患、がんなどの悪性
腫瘍に際して認められる。ビリルビンおよびその抱合体
の定量は、これらの疾患の診断、治療効果の判定に極め
て有効とされ、また最も鋭敏な肝機能検査法と考えられ
ている。(Prior art) Heme of hemoproteins such as hemoglobin that has lost its function in vivo is converted to biliverdin by opening the porphyrin ring in order to recover and reuse its iron atom, and then it is reduced. Become bilirubin. Bilirubin is poorly soluble in water and must be conjugated to excrete it in vitro. Currently, as a conjugate, a glycuronic acid conjugate, a glucose conjugate, a xylose conjugate, a conjugate "C", etc. are known. The pathological condition in which the conjugate or excretion of bilirubin is impaired due to some cause is called jaundice, and it is found in malignant tumors such as digestive system diseases such as liver, blood diseases, and cancer. The quantification of bilirubin and its conjugates is extremely effective in diagnosing these diseases and determining the therapeutic effect, and is considered to be the most sensitive liver function test method.
ところで、現在広く臨床検査法として使用されている
ヂアゾ法は、理論的にもビリルビンおよびその抱合体を
分別定量することが不可能である。By the way, the diazo method, which is widely used as a clinical test method at present, cannot theoretically separate and quantify bilirubin and its conjugate.
ビリルビンおよびその抱合体を高感度で分別定量する
ことができる最初の方法トしては、本発明者らによって
開発された高速液体クロマトグラフィー法による分析法
があり、この方法は研究室レベルで広く利用されてい
る。しかし、この方法は高価な機器を要すること、検体
の分析に１時間前後という長時間を要することなどのた
めに、臨床検査レベルでの応用は困難である。The first high-sensitivity differential quantification method for bilirubin and its conjugates was the high-performance liquid chromatography method developed by the present inventors, which is widely used at the laboratory level. It's being used. However, since this method requires expensive equipment and requires a long time of about 1 hour to analyze a sample, it is difficult to apply it at the clinical examination level.
（発明の解決しようとする問題点） そこで、遊離ビリルビンやビリルビン抱合体を短時間
にかつ高感度で多数のサンプルを分析できる方法の開発
が強く要望されている．本発明者は、遊離ビリルビンお
よびその抱合体の個々の物質にそれぞれ特異的なモノク
ローナル抗体を作製し、これを用いた免疫化学的定量法
を開発すれば、遊離ビリルビンやビリルビン抱合体を分
別定量できることに着目して、遊離ビリルビン、ビリル
ビン抱合体にそれぞれ特異的なモノクローナル抗体の作
製を試みている。(Problems to be Solved by the Invention) Therefore, there is a strong demand for the development of a method capable of analyzing a large number of samples of free bilirubin or a bilirubin conjugate in a short time with high sensitivity. The present inventor can produce a monoclonal antibody specific for each substance of free bilirubin and its conjugate, and develop an immunochemical assay using this monoclonal antibody to separate and quantify free bilirubin and a bilirubin conjugate. Focusing on, we are trying to produce monoclonal antibodies specific for free bilirubin and bilirubin conjugate.
この方法によって、基礎医学的研究面では、ビリルビ
ンの肝細胞、脳細胞などの細胞内での抱合ならびに輸送
機序などの解明に極めて有用な手段となっている。ま
た、臨床医学面では、各種疾患患者から得られた血液、
胆汁、脳脊髄液などに含まれるビリルビンおよびその抱
合体を短時間に効率よく、高い特異性と検出感度でもっ
て定量分析が可能になり、健康人についての最も鋭敏な
肝機能検査法として使用することができる物である。ま
た、このモノクローナル抗体を使用する分析法は、各種
疾患の鑑定診断、治療効果の判定などばかりでなく、そ
れらの病態の理解のための極めて有効な手段となってい
る。By this method, in the field of basic medical research, it is an extremely useful means for clarifying the conjugation of bilirubin in cells such as hepatocytes and brain cells, and the transport mechanism. In clinical medicine, blood obtained from patients with various diseases,
Bilirubin and its conjugates contained in bile, cerebrospinal fluid, etc. can be quantitatively analyzed in a short time efficiently and with high specificity and detection sensitivity, and used as the most sensitive liver function test method for healthy people. It is something that can be done. In addition, the analysis method using this monoclonal antibody has become an extremely effective means for understanding not only the diagnostic diagnosis of various diseases and the determination of therapeutic effect but also the pathological condition thereof.
従って、本発明者は、遊離ビリルビンおよびその抱合
体の個々の物質にそれぞれ特異的なモノクローナル抗体
を作製し、これを用いた免疫化学的定量法を開発すれ
ば、遊離ビリルビンやビリルビン抱合体を分別定量でき
ることに着目して、遊離ビリルビンおよびビリルビン抱
合体にそれぞれ特異的なモノクローナル抗体を作製を試
みた。しかし、これらモノクローナル抗体が認識できる
抗原には限度があり、それに応じてモノクローナル抗体
にも自ずから限度があることになる。そこで、生体内に
存在するビリルビンと形態が同じかまたはそれに極めて
類似しているビリルビン誘導体を抗原として得ること
は、これを認識できるモノクローナル抗体を産生する上
に極めて有効である。従って、かかるビリルビン誘導体
を調製することは、モノクローナル抗体によるビリルビ
ンの分析を、より効果的にかつ高感度におこなうことが
できることになる。Therefore, the present inventor produces monoclonal antibodies specific for individual substances of free bilirubin and its conjugate, and develops an immunochemical quantification method using this, to separate free bilirubin and bilirubin conjugate. Focusing on quantification, we attempted to produce monoclonal antibodies specific to free bilirubin and bilirubin conjugate. However, there is a limit to the antigens that can be recognized by these monoclonal antibodies, and accordingly, there is also a limit to the monoclonal antibodies. Therefore, obtaining a bilirubin derivative having the same form as or very similar to that of bilirubin present in the body as an antigen is extremely effective for producing a monoclonal antibody capable of recognizing this. Therefore, the preparation of such a bilirubin derivative enables the analysis of bilirubin by a monoclonal antibody to be carried out more effectively and with high sensitivity.
（問題点を解決するための手段、作用） 本発明に係るビリルビン誘導体は、一般式： BR-Z ［Ｉ］ 「式中、BRは、一般式［II］： （式中、R1およびR2は、同一もしくは異なっていて、水
素原子、グルコース残基、グルクロン酸残基またはキシ
ロース残基を意味する） で表されるビリルビン残基を意味し、 Ｚは式 −Ｘ−Y1−NH2（置換基１） （式中、Ｘは硫黄原子または酸素原子を意味し、そして
Y1は炭素原子が１〜６個の、置換基を有していてもよい
低級アルキレン基を意味する。）、 式 −Ｘ−Y1−Ｎ＝Y2−CHO （置換基２） （式中、Y2はジアルデヒド残基を意味する。）、 式 −Ｘ−Y1−Ｎ＝Y3−Ｎ−PT （置換基３） （式中、Y3は、基Y2−CH＝または二官能性架橋化合物残
基を意味し、そしてPTはタンパク質残基を意味す
る。）、または 式 −Ｘ−Y1−NH−Y3′−NH−PT （置換基４） （式中、Y3′は式Y3の還元基を意味する。） で表される残基を意味する。］ で表される。(Means and Actions for Solving Problems) The bilirubin derivative according to the present invention has a general formula: BR-Z [I] "wherein BR is a general formula [II]: (Wherein R 1 and R 2 are the same or different and each represents a hydrogen atom, a glucose residue, a glucuronic acid residue or a xylose residue), and Z represents a bilirubin residue. —X—Y 1 —NH 2 (Substituent 1) (wherein X represents a sulfur atom or an oxygen atom, and
Y1 means a lower alkylene group having 1 to 6 carbon atoms, which may have a substituent. ), Formula-X-Y1-N = Y2-CHO (Substituent 2) (In the formula, Y2 means a dialdehyde residue.), Formula-X-Y1-N = Y3-N-PT (Substituent 3) (wherein Y3 means a group Y2-CH = or a bifunctional cross-linking compound residue, and PT means a protein residue), or a formula -X-Y1-NH-Y3'-. NH-PT (Substituent 4) (wherein Y3 'represents a reducing group of formula Y3). ] Is represented.
一般式［Ｉ］において、記号Ｚの置換基１のY1で表さ
れる低級アルキレン基としては、炭素原子が１〜６個で
あって、直鎖状または分岐状の２価飽和脂肪族炭化水素
残基を意味する。なお、低級アルキレン基には、水酸基
やカルボキシル基等の置換基が存在していてもよい。か
かる低級アルキレン基の例としては、例えば、メチレン
基、エチレン基、プロピレン基、メチルプロピレン基、
ジメチルプロピレン基、ヘキシレン基、ヒドロキシエチ
レン基、カルボキシルエチレン基等が上げられる。In the general formula [I], the lower alkylene group represented by Y1 of the substituent 1 of the symbol Z has 1 to 6 carbon atoms, and is a linear or branched divalent saturated aliphatic hydrocarbon. Means a residue. The lower alkylene group may have a substituent such as a hydroxyl group or a carboxyl group. Examples of such lower alkylene groups include, for example, methylene group, ethylene group, propylene group, methylpropylene group,
Examples thereof include dimethylpropylene group, hexylene group, hydroxyethylene group and carboxylethylene group.
また、一般式［Ｉ］において、記号Ｚの置換基２のY2
で表されるジアルデヒド残基とは、置換基１のアミノ基
とシッフ塩基を形成したジアルデヒド残基を意味する。
従って、Y2で表されるジアルデヒド残基は、より具体的
には、例えば次のように表すことができる。In the general formula [I], Y2 of the substituent 2 represented by the symbol Z
The dialdehyde residue represented by means a dialdehyde residue that forms a Schiff base with the amino group of Substituent 1.
Therefore, the dialdehyde residue represented by Y2 can be more specifically represented as follows, for example.
（式中、ｎは整数を意味する。） 更に、一般式［Ｉ］において、記号Ｚの置換基３のY3
で表される二官能性架橋化合物残基とは、その二官能性
基は、その一方の官能性基が通常置換基２のアルデヒド
基とシッフ塩基を構成するか又はアミノ基と結合すると
同時に、その他方の官能性基もPTで表されるタンパク質
残基のアミノ基とシッフ塩基を構成しうるものを有する
架橋化合物の残基を意味している。 (In the formula, n means an integer.) Further, in the general formula [I], Y3 of the substituent 3 represented by the symbol Z.
The bifunctional cross-linking compound residue represented by means that the bifunctional group has one of its functional groups, which normally forms a Schiff base with the aldehyde group of the substituent 2 or bonds with an amino group, The other functional group also means the residue of a cross-linking compound having a Schiff base that can form the amino group of the protein residue represented by PT.
また置換基４は、置換基３を還元して得られた還元基
を意味している。The substituent 4 means a reducing group obtained by reducing the substituent 3.
更に置換基３および４においてPT-N＝またはPT-NH−
で表されるタンパク質は、当然のことながらその分子内
に、シッフ塩基を形成しうるアミノ基を有していなけれ
ばならない。Further, in the substituents 3 and 4, PT-N = or PT-NH-
The protein represented by must naturally have an amino group capable of forming a Schiff base in its molecule.
次に、一般式［Ｉ］で表されるビリルビン誘導体の製
造方法について簡単に説明する。Next, a method for producing the bilirubin derivative represented by the general formula [I] will be briefly described.
まず、一般式［Ｉ］で表されるビリルビン誘導体のう
ち、置換基１を有するビリルビン誘導体は、出発物質と
して使用されるビリルビン類に、例えば、システイン、
システアミン等のアミノ基とスルフヒドリル（SH）基と
を有する化合物を反応させて得ることができる。この反
応は、トルエンスルホン酸等のスルホン酸等を用いて、
酸性下で行うのが好ましい。また、この反応は、出発物
質として使用されるビリルビン類が不安定なことから室
温もしくはそれ以下の温度の条件下で行うのが好まし
い。First, among the bilirubin derivatives represented by the general formula [I], a bilirubin derivative having a substituent 1 is a bilirubin derivative used as a starting material, for example, cysteine,
It can be obtained by reacting a compound having an amino group such as cysteamine and a sulfhydryl (SH) group. This reaction uses a sulfonic acid such as toluene sulfonic acid,
It is preferably carried out under acidic conditions. Further, this reaction is preferably carried out at room temperature or lower temperature because the bilirubins used as starting materials are unstable.
この反応において出発物質として使用されるビリルビ
ン類としては、例えば、遊離ビリルビン、ビリルビン抱
合体、例えば、グルコース抱合体、キシロール抱合体、
グルクロン酸抱合体、抱合体"C"等が挙げられる。これ
らの出発物質は人体に存在するものであっても、合成し
たものであってもよい。Examples of bilirubins used as a starting material in this reaction include free bilirubin, bilirubin conjugates such as glucose conjugates, xylol conjugates,
Examples thereof include a glucuronic acid conjugate and a conjugate "C". These starting materials may be present in the human body or may be synthesized.
また一般式［Ｉ］で表されるビリルビン誘導体のう
ち、置換基２を有するビリルビン誘導体は、前記反応に
よって生成された置換基１を有するビリルビン誘導体に
ジアルデヒド化合物を作用させて得ることができる。こ
の反応によって置換基１のアミノ基は、ジアルデヒド化
合物の一方のアルデヒド基と反応してシッフ塩基を構成
する。この反応に使用されるジアルデヒド化合物は、よ
り具体的には、次のように表わすこともできる。Further, among the bilirubin derivatives represented by the general formula [I], the bilirubin derivative having the substituent 2 can be obtained by allowing a dialdehyde compound to act on the bilirubin derivative having the substituent 1 produced by the above reaction. By this reaction, the amino group of Substituent 1 reacts with one aldehyde group of the dialdehyde compound to form a Schiff base. More specifically, the dialdehyde compound used in this reaction can be represented as follows.
OHCCH2 nCHO （式中、ｎは整数を意味する。） かかるジアルデヒド化合物としては、例えば、マロンジ
アルデヒド、スクシンジアルデヒド、グルタールジアル
デヒド、アジピンジアルデヒド等が挙げられる。OHCCH 2 n CHO (In the formula, n means an integer.) Examples of such dialdehyde compounds include malondialdehyde, succindialdehyde, glutardialdehyde, adipinedialdehyde and the like.
更に、一般式［Ｉ］で表されるビリルビン誘導体のう
ち、置換基３を有するビリルビン誘導体は、前記反応で
得られた置換基２を有するビリルビン誘導体にPT-NH2で
表されるタンパク質を反応させて得ることができる。こ
のタンパク質は、抗原として作用するもので、例えば、
ヘモシアニン、アルブミン等が挙げられる。また、置換
基３を有するビリルビン誘導体は、置換基１を有するビ
リルビン誘導体に、ジアルデヒド化合物とタンパク質を
同時に反応させても得ることができる。Further, among the bilirubin derivatives represented by the general formula [I], the bilirubin derivative having the substituent 3 is obtained by reacting the bilirubin derivative having the substituent 2 obtained in the above reaction with the protein represented by PT-NH 2. You can get it. This protein acts as an antigen, for example,
Examples include hemocyanin and albumin. The bilirubin derivative having the substituent 3 can also be obtained by simultaneously reacting the bilirubin derivative having the substituent 1 with a dialdehyde compound and a protein.
更に、置換基３を有するビリルビン誘導体は、置換基
１を有するビリルビン誘導体に、二官能性架橋化合物と
タンパク質を反応させて得ることができる。この反応
は、ほぼpH7〜11で行うのが好ましい。この様な架橋化
合物の例としては、ジサクシニミジルタートレート、エ
チレングリコールビス（サクシニミジルサクシネート）
等のジサクシニミジル化合物、ビス（スルホサクシニミ
ジル）スベレート等のビススルホサクシニミジル化合物
もしくは1,5−ジフルオロ−2,4−ジニトロベンゼン等の
ジフルオロジニトロベンゼンなどが挙げられる。Further, the bilirubin derivative having the substituent 3 can be obtained by reacting the bilirubin derivative having the substituent 1 with a bifunctional crosslinking compound and a protein. The reaction is preferably carried out at approximately pH 7-11. Examples of such cross-linking compounds include disuccinimidyl tartrate, ethylene glycol bis (succinimidyl succinate)
And the like, bis (sulfosuccinimidyl) suberate and other bissulfosuccinimidyl compounds, and 1,5-difluoro-2,4-dinitrobenzene and other difluorodinitrobenzenes.
この反応で得られた置換基３を有するビリルビン誘導
体に存在するシッフ塩基は、還元して置換基４を有する
ビリルビン誘導体を得ることができる。この還元反応に
使用される還元剤としては、シッフ塩基を還元できるも
のであればいずれでもよいが、水素化物、特に水素化ほ
う素ナトリウム等が挙げられる。The Schiff base present in the bilirubin derivative having the substituent 3 obtained by this reaction can be reduced to obtain the bilirubin derivative having the substituent 4. The reducing agent used in this reduction reaction may be any agent as long as it can reduce the Schiff base, and examples thereof include hydrides, particularly sodium borohydride.
（効果） この発明に係るビリルビン誘導体は、特に遊離ビリル
ビンおよびビリルビン抱合体を人体に存在している形態
のままで特異的に認識し得るモノクローナル抗体に対す
る抗原の前駆物質もしくは抗原そのものとして機能する
ことができる。従って、この発明に係るビリルビン誘導
体は、黄疽等の病態等を診断する場合の指標となる遊離
ビリルビンやビリルビン抱合体を特異的に認識できるモ
ノクローナル抗体を製造するための抗原として特に有用
である。(Effect) The bilirubin derivative according to the present invention can function as a precursor of an antigen for a monoclonal antibody or an antigen itself which can specifically recognize free bilirubin and a bilirubin conjugate in a form existing in the human body. it can. Therefore, the bilirubin derivative according to the present invention is particularly useful as an antigen for producing a monoclonal antibody capable of specifically recognizing free bilirubin or a bilirubin conjugate, which is an index for diagnosing a pathological condition such as jaundice.
（実施例） 実施例１ ビリルビンを0.5％（w/v）システインを含有するクロ
ロホルム（1mg/ml）に溶解し、この溶液にｐ−トルエン
スルホン酸の結晶を数個添加した。この混合液を暗所に
室温で約15時間放置して、ビリルビンのエキソービニル
基にシステインのチオール基が結合したビリルビン誘導
体が得られた。(Example) Example 1 Bilirubin was dissolved in chloroform (1 mg / ml) containing 0.5% (w / v) cysteine, and several crystals of p-toluenesulfonic acid were added to this solution. This mixture was left in the dark at room temperature for about 15 hours to obtain a bilirubin derivative in which the thiol group of cysteine was bound to the exovinyl group of bilirubin.
なお、の反応には、マニット・モンティ法（Manitto
and Monti: Experientia,29,137-9,1973）の変法を使用
した。In addition, the Manit-Monti method (Manitto
and Monti: Experientia, 29 , 137-9, 1973).
実施例２ 実施例１で得られたビリルビン誘導体5mgを、0.1Mリ
ン酸緩衝液（pH6.8）1mlに溶解し、この溶液に子牛血清
アルブミン12mgを溶解した。この溶液をゆっくりと攪拌
しながら、この溶液にグルタールジアルデヒドの１％溶
液0.05mlを滴下した。この反応溶液を室温で２時間放置
し、次いで緩衝液を添加した生理食塩水各５リットルを
用いて２回４℃で一夜透析した。次に、生成した沈殿物
を４℃、20,000Gで30分間遠心分離して除去した。得ら
れたアルブミン結合ビリルビン誘導体の溶液は、使用す
るまで−80℃で保存した。Example 2 5 mg of the bilirubin derivative obtained in Example 1 was dissolved in 1 ml of 0.1 M phosphate buffer (pH 6.8), and 12 mg of calf serum albumin was dissolved in this solution. While slowly stirring this solution, 0.05 ml of a 1% solution of glutardialdehyde was added dropwise to this solution. The reaction solution was allowed to stand at room temperature for 2 hours, and then dialyzed twice with 5 liters of a physiological saline containing a buffer solution at 4 ° C. overnight. Next, the generated precipitate was removed by centrifugation at 20,000 G for 30 minutes at 4 ° C. The obtained solution of albumin-bound bilirubin derivative was stored at -80 ° C until use.
実施例３ 実施例１においてシステインの代わりにシステアミン
を使用する以外は、実施例１と同じ方法によってビリル
ビンのエキソービニル基にシステアミンのチオール基が
結合したビリルビン誘導体が得られた。Example 3 A bilirubin derivative in which a thiol group of cysteamine was bonded to an exovinyl group of bilirubin was obtained by the same method as in Example 1 except that cysteamine was used instead of cysteine in Example 1.
実施例４ 実施例３で得られたビリルビン誘導体を使用して、ア
ルブミンの代わりにカブトガニヘモシアニンを使用する
以外は実施例２の方法を用いて、ヘモシアニンが結合し
たビリルビン誘導体が得られた。Example 4 Using the bilirubin derivative obtained in Example 3, a hemocyanin-bound bilirubin derivative was obtained using the method of Example 2 except that horseshoe crab hemocyanin was used instead of albumin.
実施例５ 実施例１と同様にしてビリルビンのグルクロン酸抱合
体とシステインと反応させて、グルクロン酸抱合体のシ
ステイン付加物を得た。Example 5 The glucuronic acid conjugate of bilirubin was reacted with cysteine in the same manner as in Example 1 to obtain a cysteine adduct of the glucuronic acid conjugate.
実施例６ 実施例５で得られたビリルビン誘導体を、実施例４と
同様にしてカブトガニヘモシアニンと反応させて対応す
るシッフ塩基を得た。Example 6 The bilirubin derivative obtained in Example 5 was reacted with horseshoe crab hemocyanin in the same manner as in Example 4 to obtain the corresponding Schiff base.
このシッフ塩基を水素化ほう素ナトリウムを用いて室
温で攪拌して還元して、対応する還元型の化合物を得
た。This Schiff base was reduced by stirring with sodium borohydride at room temperature to obtain the corresponding reduced compound.
実施例７ 実施例３と同様にして、ビリルビンのグルコース抱合
体とシステアミンと反応させて、グルコース抱合体とシ
ステアミン付加物を得た。Example 7 In the same manner as in Example 3, the glucose conjugate of bilirubin was reacted with cysteamine to obtain a glucose conjugate and a cysteamine adduct.
実施例８ 実施例７で得られたシステアミン付加物を、実施例４
と同様にしてカブトガニヘモシアニンと反応させて対応
するシッフ塩基を得た。Example 8 The cysteamine adduct obtained in Example 7 was used in Example 4
The corresponding Schiff base was obtained by reacting with horseshoe crab hemocyanin in the same manner as in.
次に、このシッフ塩基を水素化ほう素ナトリウムを用
いて室温で攪拌して還元して、対応する還元型の化合物
を得た。Next, the Schiff base was reduced by stirring with sodium borohydride at room temperature to obtain a corresponding reduced compound.
実施例９ 実施例１と同様にして、ビリルビンのキシロール抱合
体とシステアミンと反応させて、キシロール抱合体とシ
ステアミン付加物を得た。Example 9 In the same manner as in Example 1, a xylol conjugate of bilirubin was reacted with cysteamine to obtain a xylol conjugate and a cysteamine adduct.
実施例10 実施例９で得られたシステアミン付加物を、実施例２
と同様にして子牛血清アルブミンと反応させて対応する
シッフ塩基を得た。Example 10 The cysteamine adduct obtained in Example 9 was used in Example 2
The corresponding Schiff base was obtained by reacting with calf serum albumin in the same manner as in.
実施例11 実施例10で得られたこのシッフ塩基を水素化ほう素ナ
トリウムを用いて室温で攪拌して還元して、対応する還
元型の化合物を得た。Example 11 The Schiff base obtained in Example 10 was reduced by stirring with sodium borohydride at room temperature to obtain the corresponding reduced compound.
Claims (1)
素原子、グルコース残基、グルクロン酸残基またはキシ
ロース残基を意味する） で表されるビリルビン残基を意味し、 Ｚは式−Ｘ−Y1−NH2 （式中、Ｘは硫黄原子または酸素原子を意味し、そして
Y1は炭素原子が１〜６個の、置換基としてヒドロキシ基
またはカルボキシル基を有していてもよい低級アルキレ
ン基を意味する。）、 式−Ｘ−Y1−Ｎ＝Y2−CHO （式中、Y2はジアルデヒド残基を意味する。）、 式−Ｘ−Y1−Ｎ＝Y3−Ｎ−PT （式中、Y3は式Y2−CH＝または一方の官能性基が上記置
換基Y2とシッフ塩基を構成するかもしくはアミノ酸と結
合し、他方の官能基が下記PTで表されるタンパク質残基
のアミノ酸とシッフ塩基を構成している二官能性架橋化
合物残基を意味し、そしてPTはタンパク質残基を意味す
る。）、または 式−Ｘ−Y1−Ｎ＝Y3′−NH−PT （式中、Y3′は式Y3の還元基を意味する。） で表される残基を意味する。］ で表されることを特徴とするビリルビン誘導体。1. A general formula: BR-Z [I] [wherein: BR is a general formula [II]: (Wherein R 1 and R 2 are the same or different and each represents a hydrogen atom, a glucose residue, a glucuronic acid residue or a xylose residue), and Z represents a bilirubin residue. —X—Y 1 —NH 2 (wherein X represents a sulfur atom or an oxygen atom, and
Y1 means a lower alkylene group having 1 to 6 carbon atoms, which may have a hydroxy group or a carboxyl group as a substituent. ), Formula-X-Y1-N = Y2-CHO (In the formula, Y2 means a dialdehyde residue.), Formula-X-Y1-N = Y3-N-PT (In formula, Y3 is Formula Y2. -CH = or one functional group constitutes a Schiff base with the above-mentioned substituent Y2 or binds to an amino acid, and the other functional group constitutes a Schiff base with an amino acid of a protein residue represented by PT below. A difunctional cross-linking compound residue, and PT means a protein residue), or a formula -X-Y1-N = Y3'-NH-PT, where Y3 'is a reduction of formula Y3. Means a group.) Means a residue represented by. ] The bilirubin derivative characterized by being represented by these.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10549687A JPH0830059B2 (en) | 1987-04-27 | 1987-04-27 | Bilirubin derivative |
| AU10424/88A AU1042488A (en) | 1986-12-20 | 1987-12-19 | Bilirubin antigen, monoclonal antibody therefor, process for their preparation, and their use |
| EP19880900121 EP0307476A4 (en) | 1986-12-20 | 1987-12-19 | Bilirubin antigen, monoclonal antibody therefor, process for their preparation, and their use |
| PCT/JP1987/000999 WO1988004670A1 (en) | 1986-12-20 | 1987-12-19 | Bilirubin antigen, monoclonal antibody therefor, process for their preparation, and their use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10549687A JPH0830059B2 (en) | 1987-04-27 | 1987-04-27 | Bilirubin derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63267757A JPS63267757A (en) | 1988-11-04 |
| JPH0830059B2 true JPH0830059B2 (en) | 1996-03-27 |
Family
ID=14409206
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10549687A Expired - Lifetime JPH0830059B2 (en) | 1986-12-20 | 1987-04-27 | Bilirubin derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0830059B2 (en) |
-
1987
- 1987-04-27 JP JP10549687A patent/JPH0830059B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| Ｅｘｐｅｒｉｅｎｔｉａ，２８（４），３７９−８０（１９７２） |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63267757A (en) | 1988-11-04 |
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