JPS6149624B2 - - Google Patents
Info
- Publication number
- JPS6149624B2 JPS6149624B2 JP56153796A JP15379681A JPS6149624B2 JP S6149624 B2 JPS6149624 B2 JP S6149624B2 JP 56153796 A JP56153796 A JP 56153796A JP 15379681 A JP15379681 A JP 15379681A JP S6149624 B2 JPS6149624 B2 JP S6149624B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- dacm
- labeled
- labeled antibody
- mercaptoethanol
- 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
Links
- ADEORFBTPGKHRP-UHFFFAOYSA-N 1-[7-(dimethylamino)-4-methyl-2-oxochromen-3-yl]pyrrole-2,5-dione Chemical compound O=C1OC2=CC(N(C)C)=CC=C2C(C)=C1N1C(=O)C=CC1=O ADEORFBTPGKHRP-UHFFFAOYSA-N 0.000 claims description 41
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 34
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 5
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims description 2
- PMUNIMVZCACZBB-UHFFFAOYSA-N 2-hydroxyethylazanium;chloride Chemical compound Cl.NCCO PMUNIMVZCACZBB-UHFFFAOYSA-N 0.000 claims 1
- -1 7-dimethylamino-4-methylcoumarinyl Chemical group 0.000 claims 1
- 229940073579 ethanolamine hydrochloride Drugs 0.000 claims 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N monoethanolamine hydrochloride Natural products NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 18
- 239000000126 substance Substances 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 238000005562 fading Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000009871 nonspecific binding Effects 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- XFZYOTCYWNADMQ-UHFFFAOYSA-N 2-amino-1-sulfanylethanol;hydrochloride Chemical compound Cl.NCC(O)S XFZYOTCYWNADMQ-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 238000009739 binding Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000000760 immunoelectrophoresis Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 1
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 1
- 206010018372 Glomerulonephritis membranous Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate Chemical compound [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 210000001914 gastric parietal cell Anatomy 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 201000008350 membranous glomerulonephritis Diseases 0.000 description 1
- 231100000855 membranous nephropathy Toxicity 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
【発明の詳細な説明】
本発明は、新規な螢光標識抗体及びその製造方
法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel fluorescently labeled antibody and a method for producing the same.
螢光物質で標識した抗体は、各種生体組識にお
ける生物学的活性分子の検出に広く用いられてい
る。 Antibodies labeled with fluorescent substances are widely used to detect biologically active molecules in various biological tissues.
標識用螢光物質としては、主にフルオレセイン
シアネート及びその誘導体が多用されているが、
これらを抗体と結合させるためには、複雑な手技
と長時間を必要とするほか、得られた螢光標識抗
体が非特異的な反応を示すことから、特異的に反
応する螢光標識抗体を得るために、更に繁雑な操
作を必要とするなど多くの問題点がある。 Fluorescein cyanate and its derivatives are mainly used as fluorescent substances for labeling.
In order to combine these with antibodies, complex procedures and a long time are required, and the resulting fluorescently labeled antibodies exhibit nonspecific reactions. There are many problems, such as the need for more complicated operations.
抗体には免疫グロブリン（以下Igと略記する）
Ｇ、IgA、IgM、IgD及びIgEの５種の型があり、
それらは構造上２つのヘビーチエイン（Ｈ鎖）と
２つのライトチエイン（Ｌ鎖）を基本単位として
いる。Ｈ鎖とＬ鎖は相互にジサルフアイド結合
（−Ｓ−Ｓ−）により結合し、Ｈ鎖同士も同様に
ジサルフアイド結合により結合しているが、この
結合は２−メルカプトエタノール又は２−メルカ
プトエタノールアミン塩酸塩により容易に切断さ
れ、それぞれが−SH基となる。次いで中性の条
件下で２−メルカプトエタノール又は２−メルカ
プトエタノールアミン塩酸塩を除去すると、再び
ジサルフアイド結合が形成され、もとの抗体とし
ての活性を示す。この際に添加する２−メルカプ
トエタノール又は２−メルカプトエタノールアミ
ン塩酸塩の濃度を変えることによつて、切断され
るジサルフアイド結合の数を変えることができ、
また切断後に生じた−SH基の水素を他の物質で
置換しても、添加した２−メルカプトエタノール
又は２−メルカプトエタノールアミン塩酸塩を除
去すると、再び分子結合によつて安定な型の抗体
となることが知られている。 Antibodies include immunoglobulin (hereinafter abbreviated as Ig)
There are 5 types: G, IgA, IgM, IgD and IgE.
Their basic unit is two heavy chains (H chains) and two light chains (L chains). H chains and L chains are bonded to each other by disulfide bonds (-S-S-), and H chains are also bonded to each other by disulfide bonds, but this bond is formed by 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride. It is easily cleaved by salt, and each becomes a -SH group. Then, when 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride is removed under neutral conditions, disulfide bonds are formed again, and the antibody exhibits its original activity. By changing the concentration of 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride added at this time, the number of disulfide bonds to be cleaved can be changed,
In addition, even if the hydrogen of the -SH group generated after cleavage is replaced with another substance, when the added 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride is removed, a stable form of the antibody is restored by molecular bonding. It is known that
本発明者らは、以上の事実に着目し、Ｎ−（７
−ジメチルアミノ−４−メチルクマリニル）マレ
イミド（DACM）を抗体のジサルフアイド結合
切断後に生じた−SH基に結合させて螢光標識抗
体を製造した。そしてこの標識抗体が、現在広く
用いられているフルオレセインイソチオシアネー
ト（FITC）で標識した抗体（以下FITC標識抗
体と略記する）に比べて非特異的染色が少なく、
かつ螢光の退色も少ないことを見出して本発明を
完成した。 The present inventors paid attention to the above facts and made N-(7
A fluorescently labeled antibody was prepared by binding -dimethylamino-4-methylcoumarinyl)maleimide (DACM) to the -SH group generated after cleavage of the disulfide bond of the antibody. This labeled antibody has less non-specific staining than the currently widely used antibody labeled with fluorescein isothiocyanate (FITC) (hereinafter abbreviated as FITC-labeled antibody).
The present invention was completed based on the discovery that fading of the fluorescent light was also minimal.
本発明は、DACMで標識した螢光標識抗体
（DACM標識抗体と略記する）、ならびに抗体を
２−メルカプトエタノール又は２−メルカプトエ
タノールアミン塩酸塩で処理し、次いでDACM
と反応させたのち、２−メルカプトエタノール又
は２−メルカプトエタノールアミン塩酸塩を除去
することを特徴とする、前記の標識抗体の製造方
法である。 The present invention involves treating a fluorescently labeled antibody labeled with DACM (abbreviated as DACM-labeled antibody) and the antibody with 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride, and then treating the antibody with DACM.
The above method for producing a labeled antibody is characterized in that 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride is removed after reacting with the labeled antibody.
本発明においてDACMは、チオール基と結合
することにより螢光を発する化合物で、チオール
基の検出用試薬として知られている（特公昭51−
13035号公報参照）。また抗体としてはIgG、
IgA、IgM、IgD及びIgEを用いることができる。 In the present invention, DACM is a compound that emits fluorescence when combined with a thiol group, and is known as a reagent for detecting a thiol group (Japanese Patent Publication No. 51-1999).
(See Publication No. 13035). Also, as antibodies, IgG,
IgA, IgM, IgD and IgE can be used.
本発明のDACM標識抗体を製造するに際して
は、まず抗体を２−メルカプトエタノール又は２
−メルカプトエタノールアミン塩酸塩で処理す
る。抗体としては、通常は抗血清の抗体を含む分
画を用い、これをPH6.5〜8.5の緩衝液に溶解す
る。緩衝液としては、例えばトリス塩酸塩緩衝液
（PH8.2）、燐酸緩衝液（PH8.2）等が用いられる。
抗体を含む分画の濃度は0.5〜１％（Ｗ／Ｖ）が
好ましい。 When producing the DACM-labeled antibody of the present invention, first the antibody is mixed with 2-mercaptoethanol or 2-mercaptoethanol.
- treatment with mercaptoethanolamine hydrochloride. As the antibody, an antibody-containing fraction of antiserum is usually used, and this is dissolved in a buffer solution with a pH of 6.5 to 8.5. As the buffer, for example, Tris hydrochloride buffer (PH8.2), phosphate buffer (PH8.2), etc. are used.
The concentration of the antibody-containing fraction is preferably 0.5 to 1% (W/V).
次いでこの抗体溶液に、２−メルカプトエタノ
ール又は２−メチルメルカプトエタオールアミン
塩酸塩を添加する。これら化合物の添加量は、最
終濃度が0.25〜4mMになるように調整すること
が好ましい。この混合液を室温に約１時間放置す
ると反応が終了する。 2-mercaptoethanol or 2-methylmercaptoethanolamine hydrochloride is then added to this antibody solution. The amount of these compounds added is preferably adjusted so that the final concentration is 0.25 to 4 mM. The reaction is completed when this mixture is left at room temperature for about 1 hour.
こうして得られた混合液にDACMを添加し、
更に約１時間室温で反応させる。DACMの添加
量は、２−メルカプトエタノール又は２−メルカ
プトエタノールアミン塩酸塩と等モル量程度が好
ましい。 Adding DACM to the mixture thus obtained,
The reaction is further allowed to proceed for about 1 hour at room temperature. The amount of DACM added is preferably approximately equimolar to 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride.
反応終了後に２−メルカプトエタノール又は２
−メルカプトエタノールアミン塩酸塩を除去する
には、例えば反応液を中性の緩衝液に対して透析
する。中性の緩衝液としては、例えば燐酸ソーダ
緩衝液（PH7.4）等があげられる。この溶液をゲ
ル過して蛋白分画を採取すると、本発明の
DACM標識抗体が得られる。 After the reaction is complete, add 2-mercaptoethanol or 2-mercaptoethanol.
- To remove mercaptoethanolamine hydrochloride, for example, the reaction solution is dialyzed against a neutral buffer. Examples of the neutral buffer include sodium phosphate buffer (PH7.4). When this solution is gel-filtered and the protein fraction is collected, the present invention
A DACM-labeled antibody is obtained.
この分画がDACMにより螢光標識されている
ことは、第１図に示すように最大吸収波長が
395nmを示し、468nmに放出スペクトルがみられ
ることから確認される。またこの分画が目的の型
の抗体であることは、二元拡散ゲル内沈降反応又
は免疫電気泳動法により確認され、同時に反応す
る抗原とのみ反応することも確認できる。 The fact that this fraction is fluorescently labeled by DACM means that the maximum absorption wavelength is
395nm, and is confirmed by the emission spectrum seen at 468nm. Furthermore, it is confirmed that this fraction is the desired type of antibody by a binary diffusion gel precipitation reaction or immunoelectrophoresis, and it can also be confirmed that the fraction reacts only with the antigen with which it reacts.
本発明のDACM標識抗体は、非特異的染色及
び螢光の褪色が少ないため、螢光顕微鏡を用いた
螢光抗体法による生体組識中の特異的抗原物質検
出用螢光標識抗体として極めて好適である。 The DACM-labeled antibody of the present invention is extremely suitable as a fluorescently labeled antibody for detecting specific antigenic substances in biological tissues by fluorescent antibody method using a fluorescent microscope because it has little nonspecific staining and fading of fluorescence. It is.
抗体に結合する螢光色素量は、通常はＦ／Ｐ値
（螢光色素のモル濃度／蛋白質のモル濃度）とし
て表わされる。FITC標識抗体ではＦ／Ｐ値が大
きくなる程、電気泳動にかけた際の移動度が大き
くなるが、これはFITCが抗体に結合することに
より、標識抗体が負の荷電を帯びるためである。
このような標識抗体を用いて生体組織中の特異抗
原物質を染色すると、標識抗体が負の電荷を帯び
ていることから特異抗原物質のみならず、他の正
の電荷を帯びた部分とも結合して、非特異的螢光
像を示すこととなる。したがつて特異的螢光像を
得るためには、適当なＦ／Ｐ値（１〜２）の
FITC標識抗体を調製する必要があるが、これに
は繁雑な操作が必要となる。 The amount of fluorescent dye that binds to an antibody is usually expressed as an F/P value (fluorescent dye molar concentration/protein molar concentration). The larger the F/P value of an FITC-labeled antibody, the greater its mobility during electrophoresis, and this is because the labeled antibody becomes negatively charged when FITC binds to the antibody.
When such a labeled antibody is used to stain a specific antigen substance in living tissue, since the labeled antibody is negatively charged, it binds not only to the specific antigen substance but also to other positively charged parts. This results in a non-specific fluorescence image. Therefore, in order to obtain a specific fluorescence image, an appropriate F/P value (1 to 2) must be set.
It is necessary to prepare FITC-labeled antibodies, but this requires complicated operations.
これに対し、本発明のDACM標識抗体は、後
記の実験例４に示すように、DACMが抗体に結
合しても荷電に変化が生じないので、Ｆ／Ｐ値の
大きい抗体を用いても、生体組織中の特異抗原物
質のみと結合するため、特異的螢光像を得ること
ができる。 In contrast, with the DACM-labeled antibody of the present invention, as shown in Experimental Example 4 below, the charge does not change even when DACM binds to the antibody, so even if an antibody with a large F/P value is used, Because it binds only to specific antigenic substances in living tissues, specific fluorescent images can be obtained.
生体組織中の特異抗原物質を、螢光標識抗体を
用いて染色する際、特異抗原物質が極微量である
場合は螢光量の多い標識抗体が必要であり、また
逆に特異抗原物質が比較的多い場合は螢光量の少
い標識抗体が用いられる。本発明の製造方法によ
れば任意のＦ／Ｐ値のDACM標識抗体を容易に
製造することができる。 When staining a specific antigen substance in living tissue using a fluorescently labeled antibody, if the specific antigen substance is in an extremely small amount, a labeled antibody with a large amount of fluorescence is required; If the amount of fluorescent light is large, a labeled antibody with a low amount of fluorescence is used. According to the production method of the present invention, a DACM-labeled antibody with any F/P value can be easily produced.
本発明方法では、２−メルカプトエタノール又
は２−メルカプトエタノールアミン塩酸塩の濃度
を変えることにより、抗体のジサルフアイド結合
の切断後に生ずる−SH基の数を変えることがで
きるため、−SH基に結合するDACMの量も変える
ことができる。すなわち、２−メルカプトエタノ
ール又は２−メルカプトエタノールアミン塩酸塩
の濃度を変えることにより螢光標識抗体のＦ／Ｐ
値を調節できる。 In the method of the present invention, by changing the concentration of 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride, the number of -SH groups generated after cleavage of the disulfide bond of the antibody can be changed. The amount of DACM can also be varied. That is, by changing the concentration of 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride, the F/P of the fluorescently labeled antibody can be adjusted.
You can adjust the value.
本発明方法により得られるDACM標識抗体の
Ｆ／Ｐ値と、DACMの添加濃度との間には、第
２図に示すような一定の関係のあることが認めら
れた。この図から明らかなように、本発明方法に
よれば、DACM濃度を変えることにより、任意
のＦ／Ｐ値を有するDACM標識抗体を得ること
ができる。 It was found that there was a certain relationship between the F/P value of the DACM-labeled antibody obtained by the method of the present invention and the concentration of DACM added, as shown in FIG. As is clear from this figure, according to the method of the present invention, a DACM-labeled antibody having an arbitrary F/P value can be obtained by changing the DACM concentration.
実施例
抗ヒトIgGウサギ血清IgG（16mg／ml、DAKO
−イムノグロブリンズａ／Ｓ社製）１mlに同量の
1.1Mトリス塩酸緩衝液（PH8.2）を加え、0.8％
（Ｗ／Ｖ）抗ヒトIgGウサギ血清IgGの0.55Mトリ
ス塩酸緩衝液（PH8.2）を調製する。２−メルカ
プトエタノール（東京化成社製）37μを再蒸留
水５mlに溶解して100mM溶液を調製し、その80
μｌを前記の0.8％抗ヒトIgGウサギ血清IgG緩衝
溶液２mlに加え、この混合液を室温で１時間反応
させる。Example Anti-human IgG rabbit serum IgG (16 mg/ml, DAKO
- Immunoglobulins A/S) 1 ml of the same amount
Add 1.1M Tris-HCl buffer (PH8.2), 0.8%
(W/V) Prepare anti-human IgG rabbit serum IgG in 0.55 M Tris-HCl buffer (PH8.2). Dissolve 37μ of 2-mercaptoethanol (manufactured by Tokyo Kasei Co., Ltd.) in 5ml of double-distilled water to prepare a 100mM solution.
μl is added to 2 ml of the above 0.8% anti-human IgG rabbit serum IgG buffer solution, and this mixture is allowed to react at room temperature for 1 hour.
次いでDACM（和光純薬社製）10mgを3.35ml
のアセトンに溶解して最終濃度10mM溶液を調製
し、この溶液0.8mlを前記の混合液に加え、更に
室温で１時間反応させる。この反応溶液をシーム
レスセルロース透析膜チユーブ（ヴイスキング社
製、８／32型）に入れ、４℃で12時間、大量の
5mM燐酸ソーダ緩衝液（PH7.4）に対して透析
し、透析後セルローズチユーブ内の溶液をセフア
デツクスＧ−50（フアルマシア社製ゲル過剤）
を充填したカラム（フアルマシア社製、C26／40
型）により、0.9％食塩を含む5mM燐酸ソーダ緩
衝液（PH7.4）を用いてゲル過し、最初に流出
してくる蛋白分画を採取する。この蛋白分画を限
外過機（アミコン社製、202型）によりダイア
フローメンブレン（アミコン社製、PM10型）を
用いて任意の蛋白濃度に濃縮し、目的のDACM
標識抗体の蛋白分画を得る。 Next, add 3.35ml of 10mg of DACM (manufactured by Wako Pure Chemical Industries)
to prepare a final concentration of 10mM solution, add 0.8ml of this solution to the above mixture, and further react for 1 hour at room temperature. This reaction solution was placed in a seamless cellulose dialysis membrane tube (manufactured by VISKING, model 8/32), and a large amount of
Dialyzed against 5mM sodium phosphate buffer (PH7.4), and after dialysis, the solution in the cellulose tube was filtered with Cephadex G-50 (gelling agent manufactured by Pharmacia).
Column packed with (manufactured by Pharmacia, C26/40
gel filtration using 5mM sodium phosphate buffer (PH7.4) containing 0.9% salt, and collect the protein fraction that flows out first. This protein fraction is concentrated to a desired protein concentration using a diaflow membrane (PM10 model, Amicon Corporation) using an ultrafilter (manufactured by Amicon Corporation, Model 202), and the desired DACM
Obtain the protein fraction of the labeled antibody.
この蛋白分画がDACMにより螢光標識されて
いることは、吸収スペクトラムの最大波長が
395nmを示し、放出スペクトラムの最大波長が
468nmを示すこと、及び寒天平板電気泳動により
蛋白泳動部分と螢光発色部分とが一致することか
ら確認された。またこの蛋白が抗ヒトIgGウサギ
血清IgGであり、対応するIgGとのみ反応するこ
とは、ヒト血清及びヒトIgGを用いた二元拡散ゲ
ル内沈降反応及び免疫電気泳動法により得られた
沈降線が単一バンドを示すことから確認された。 The fact that this protein fraction is fluorescently labeled by DACM means that the maximum wavelength of the absorption spectrum is
395nm, and the maximum wavelength of the emission spectrum is
This was confirmed by the fact that the wavelength was 468 nm and that the protein migration area and the fluorescent area coincided with each other by agar plate electrophoresis. Furthermore, this protein is anti-human IgG rabbit serum IgG, and the fact that it reacts only with the corresponding IgG means that the precipitation line obtained by binary diffusion gel precipitation reaction and immunoelectrophoresis using human serum and human IgG shows that the protein reacts only with the corresponding IgG. It was confirmed by showing a single band.
実験例 １
Ｂ−リンパ球は、その表面にIgGを有すること
が知られている。実施例と同様の方法で製造した
DACM標識抗IgG抗体（Ｆ／Ｐ＝2.3）を用いて
正常人末梢血リンパ球を染色して蛍光顕微鏡で観
察したところ、細胞表面に顆粒状の蛍光が見ら
れ、Ｂ−リンパ球が特異的に染色されていること
が確認された。Experimental Example 1 B-lymphocytes are known to have IgG on their surface. Manufactured in the same manner as in the example
When normal human peripheral blood lymphocytes were stained with DACM-labeled anti-IgG antibody (F/P = 2.3) and observed under a fluorescence microscope, granular fluorescence was seen on the cell surface, indicating that B-lymphocytes were specific. It was confirmed that it was stained.
実験例 ２
実験例１と同じDACM標識抗IgG抗体を用い、
IgGを含んだ免疫複合体の沈着物が表面に存在す
る膜性腎症の糸球体を染色したところ、塊状の沈
着物が蛍光顕微鏡で明瞭に確認できた。Experimental Example 2 Using the same DACM-labeled anti-IgG antibody as in Experimental Example 1,
When we stained a glomerulus with membranous nephropathy, which has IgG-containing immune complex deposits on its surface, the lump-like deposits were clearly visible under a fluorescence microscope.
実験例 ３
抗糸粒体抗体は原発性胆汁性肝硬変症の患者の
血中に見られることが知られている。この抗体を
ネズミ胃壁細胞で吸収し、実験例１と同じ
DACM標識抗IgG抗体を用い、免疫蛍光間接法に
より観察したところ、抗糸粒体抗体が確認され
た。Experimental Example 3 Anti-globulin antibodies are known to be found in the blood of patients with primary biliary cirrhosis. This antibody was absorbed by rat gastric parietal cells, and the same procedure as in Experiment 1 was carried out.
When observed by indirect immunofluorescence using a DACM-labeled anti-IgG antibody, anti-globular antibodies were confirmed.
以上の実験から本発明のDACM標識抗体が対
応する抗原に対し特異的結合反応を示すことが確
認された。 The above experiments confirmed that the DACM-labeled antibody of the present invention exhibits a specific binding reaction to the corresponding antigen.
実験例 ４
蛍光抗体法に用いる標識抗体には、非特異的結
合性の少ないものが要求される。そこで通常の蛍
光抗体法に用いられるFITC標識抗体と本発明の
DACM標識抗体とを用いて非特異的結合性の検
討を行つた。Experimental Example 4 The labeled antibody used in the fluorescent antibody method is required to have low non-specific binding. Therefore, the FITC-labeled antibody used in ordinary fluorescent antibody method and the present invention
Nonspecific binding was investigated using DACM-labeled antibodies.
FITC標識抗ヒトIgGウサギ血清IgG（Ｆ／Ｐ＝
2.3）、DACM標識抗ヒトIgGウサギ血清IgG
（Ｆ／Ｐ＝2.3）および未標識抗ヒトIgGウサギ血
清IgGを、それぞれ電気泳動にかけた結果、その
移動度はFITCを標識したものは−0.88×10-5
cm2／Volt／秒、DACMを標識したものは−1.00×
10-5cm2／VOlt／秒を示し、また未標識のものは
DACMを標識したものと同じ値を示した。 FITC-labeled anti-human IgG rabbit serum IgG (F/P=
2.3), DACM labeled anti-human IgG rabbit serum IgG
(F/P=2.3) and unlabeled anti-human IgG rabbit serum IgG were subjected to electrophoresis, and the mobility of the FITC-labeled one was -0.88×10 -5
cm 2 /Volt/sec, DACM labeled −1.00×
10 -5 cm 2 /VOlt/sec, and the unlabeled ones are
It showed the same value as that labeled with DACM.
FITC標識抗体ではFITCが抗体に結合するこ
とにより、負の荷電を帯びるため、Ｆ／Ｐ値が大
きくなる程、電気泳動による陽極方向への移動度
が大きくなることが知られている。このことは本
実験例４において、FITC標識抗ヒトIgGウサギ
血清IgGが、未標識のものに比較して陰極側への
移動度が少なかつたことから確認できた。FITC
が標識された結果、負の荷電を帯びた標識抗体
は、正の荷電を帯びる生体組織標本に対し非特異
的結合性を示すため、できるだけ負の荷電の少な
い、換言すればＦ／Ｐ値の小さい（２以下が望ま
しい）FITC標識抗体が必要とされている。一
方、本実験例４で用いたDACM標識抗ヒトIgGウ
サギ血清IgGは、Ｆ／Ｐ値が2.3であるにもかかわ
らず、未標識のそれと同値の電気泳動移動度を示
した。これはDACMが抗体に結合しても荷電に
変化を与えないことを意味する。このことから負
の荷電を帯びない本発明のDACM標識抗体は、
負の荷電を帯びるFITC標識抗体に比べて、生体
組織標本に対する非特異的結合性のないことが確
認された。 It is known that FITC-labeled antibodies become negatively charged when FITC binds to the antibody, and therefore, the larger the F/P value, the greater the electrophoretic mobility toward the anode. This was confirmed in Experimental Example 4, since the FITC-labeled anti-human IgG rabbit serum IgG had a lower mobility toward the cathode than the unlabeled one. FITC
The labeled antibody, which is negatively charged as a result of labeling, exhibits non-specific binding to positively charged biological tissue specimens. Small (preferably 2 or less) FITC-labeled antibodies are needed. On the other hand, the DACM-labeled anti-human IgG rabbit serum IgG used in Experimental Example 4 showed the same electrophoretic mobility as that of unlabeled IgG, although the F/P value was 2.3. This means that when DACM binds to antibodies, the charge does not change. Therefore, the DACM-labeled antibody of the present invention, which is not negatively charged,
It was confirmed that there is no non-specific binding to biological tissue specimens compared to negatively charged FITC-labeled antibodies.
実験例 ５
FITC標識抗体と本発明のDACM標識抗体を用
いて、紫外線に対する抵抗性を調べた。Experimental Example 5 Resistance to ultraviolet rays was investigated using an FITC-labeled antibody and a DACM-labeled antibody of the present invention.
同じＦ／Ｐ値を示すFITC標識抗体及びDACM
標識抗体を波長395nm及び470nmの光線で暴露
し、蛍光の消退を蛍光光度計（日立製作所製分光
光度計MPF−３型）により測定した。その結果
を第３図に示す。 FITC-labeled antibody and DACM showing the same F/P value
The labeled antibody was exposed to light having wavelengths of 395 nm and 470 nm, and the extinction of fluorescence was measured using a fluorometer (Spectrophotometer Model MPF-3 manufactured by Hitachi, Ltd.). The results are shown in FIG.
この図から明らかなように、本発明のDACM
標識抗体は蛍光の褪色が非常に少ないので、蛍光
顕微鏡をを用いる蛍光抗体法による生体組織中の
特異抗原物質検出用蛍光標識抗体として極めて有
利である。 As is clear from this figure, the DACM of the present invention
Since the labeled antibody exhibits very little fading of fluorescence, it is extremely advantageous as a fluorescently labeled antibody for detecting specific antigen substances in biological tissues by the fluorescent antibody method using a fluorescence microscope.
第１図は本発明のDACM標識抗体の励起スペ
クトラム及び蛍光スペクトラム、第２図は本発明
の製造方法によりDACMの添加量を変えた場合
の抗体結合蛍光色素量比（Ｆ／Ｐ）を示すグラ
フ、第３図は励起光照射によるDACM標識抗体
とFITC標識抗体の蛍光の褪色速度を示すグラフ
である。
Figure 1 is a graph showing the excitation spectrum and fluorescence spectrum of the DACM-labeled antibody of the present invention, and Figure 2 is a graph showing the antibody-bound fluorescent dye amount ratio (F/P) when the amount of DACM added is changed according to the production method of the present invention. , FIG. 3 is a graph showing the fading rate of fluorescence of DACM-labeled antibody and FITC-labeled antibody due to excitation light irradiation.
Claims (1)
リニル）マレイミドで標識した螢光標識抗体。 ２ 抗体を２−メルカプトエタレール又は２−メ
ルカプトエタノールアミン塩酸塩で処理し、次い
でＮ−（７−ジメチルアミノ−４−メチルクマリ
ニル）マレイミドと反応させたのち、２−メルカ
プトエタノール又は２−メルカプトエタノールア
ミン塩酸塩を除去することを特徴とする、Ｎ−
（７−ジメチルアミノ−４−メチルクマリニル）
マレイミドで標識した螢光標識抗体の製造方法。[Scope of Claims] A fluorescently labeled antibody labeled with 1 N-(7-dimethylamino-4-methylcoumarinyl)maleimide. 2 Antibodies were treated with 2-mercaptoethanol or 2-mercaptoethanolamine hydrochloride, then reacted with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, and then treated with 2-mercaptoethanol or 2-mercaptoethanolamine. N-, characterized in that it removes ethanolamine hydrochloride.
(7-dimethylamino-4-methylcoumarinyl)
A method for producing a fluorescently labeled antibody labeled with maleimide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15379681A JPS5855861A (en) | 1981-09-30 | 1981-09-30 | New fluorescent labeled antibody and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15379681A JPS5855861A (en) | 1981-09-30 | 1981-09-30 | New fluorescent labeled antibody and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5855861A JPS5855861A (en) | 1983-04-02 |
| JPS6149624B2 true JPS6149624B2 (en) | 1986-10-30 |
Family
ID=15570311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15379681A Granted JPS5855861A (en) | 1981-09-30 | 1981-09-30 | New fluorescent labeled antibody and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5855861A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016129444A1 (en) * | 2015-02-12 | 2016-08-18 | コニカミノルタ株式会社 | Antibody-conjugated integrated phosphor nanoparticles, method for manufacturing antibody-conjugated integrated phosphor nanoparticles, and immunostaining kit |
| US11105807B2 (en) | 2015-09-28 | 2021-08-31 | Konica Minolta, Inc. | Method for estimating pathological tissue diagnosis result (Gleason score) of prostate cancer |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6432170A (en) * | 1987-07-29 | 1989-02-02 | Agency Ind Science Techn | Production of novel fluorescent labeled antibody |
| US7563891B2 (en) * | 2004-05-21 | 2009-07-21 | Becton, Dickinson & Company | Long wavelength thiol-reactive fluorophores |
| US20200408688A1 (en) * | 2018-03-23 | 2020-12-31 | Konica Minolta, Inc. | Labeled antibody dispersion liquid and kit for spfs |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5113035A (en) * | 1974-07-23 | 1976-02-02 | Mitsubishi Heavy Ind Ltd | Nainenkikanno jidontensochi |
| JPS53124682A (en) * | 1977-04-08 | 1978-10-31 | Asahi Chem Ind Co Ltd | Preparation of complex of enzyme and bioactive substance and its use |
-
1981
- 1981-09-30 JP JP15379681A patent/JPS5855861A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5113035A (en) * | 1974-07-23 | 1976-02-02 | Mitsubishi Heavy Ind Ltd | Nainenkikanno jidontensochi |
| JPS53124682A (en) * | 1977-04-08 | 1978-10-31 | Asahi Chem Ind Co Ltd | Preparation of complex of enzyme and bioactive substance and its use |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016129444A1 (en) * | 2015-02-12 | 2016-08-18 | コニカミノルタ株式会社 | Antibody-conjugated integrated phosphor nanoparticles, method for manufacturing antibody-conjugated integrated phosphor nanoparticles, and immunostaining kit |
| JPWO2016129444A1 (en) * | 2015-02-12 | 2017-11-24 | コニカミノルタ株式会社 | Antibody-binding phosphor-integrated nanoparticles, method for producing antibody-binding phosphor-integrated nanoparticles, and immunostaining kit |
| US11105807B2 (en) | 2015-09-28 | 2021-08-31 | Konica Minolta, Inc. | Method for estimating pathological tissue diagnosis result (Gleason score) of prostate cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5855861A (en) | 1983-04-02 |
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