JPS60135765A - Method for inspecting contraction of cancer - Google Patents

Method for inspecting contraction of cancer

Info

Publication number
JPS60135765A
JPS60135765A JP24345383A JP24345383A JPS60135765A JP S60135765 A JPS60135765 A JP S60135765A JP 24345383 A JP24345383 A JP 24345383A JP 24345383 A JP24345383 A JP 24345383A JP S60135765 A JPS60135765 A JP S60135765A
Authority
JP
Japan
Prior art keywords
lymph cells
hematoporphyrin
lymphocytes
cancer
cells
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.)
Granted
Application number
JP24345383A
Other languages
Japanese (ja)
Other versions
JPH035551B2 (en
Inventor
Masaaki Aoyama
正明 青山
Mitsuharu Itabashi
板橋　光春
Masahiro Kono
雅弘 河野
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SUPESHIARU REFUARENSU LAB KK
Jeol Ltd
Original Assignee
SUPESHIARU REFUARENSU LAB KK
Jeol Ltd
Nihon Denshi KK
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by SUPESHIARU REFUARENSU LAB KK, Jeol Ltd, Nihon Denshi KK filed Critical SUPESHIARU REFUARENSU LAB KK
Priority to JP24345383A priority Critical patent/JPS60135765A/en
Priority to US06/608,996 priority patent/US4696905A/en
Priority to DE8484105337T priority patent/DE3483493D1/en
Priority to DE198484105337T priority patent/DE125651T1/en
Priority to EP84105337A priority patent/EP0125651B1/en
Publication of JPS60135765A publication Critical patent/JPS60135765A/en
Publication of JPH035551B2 publication Critical patent/JPH035551B2/ja
Granted legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/60—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using electron paramagnetic resonance

Landscapes

  • Physics & Mathematics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

PURPOSE:To enable exact an indentification test by adding a hematoporphyrin deriv. to the lymph cells from a person to be tested, separating only the lymph cells after incubation and measuring quantitatively the quantity of the radicals included in the separated lymph cells by using an electron spin resonator. CONSTITUTION:Only the lumph cells are taken out of the blood drawn from a patient and is mixed with, for example, 50mul hematoporphyrin driv. of 0.005mol and the mixture is incubated for about 10min at 37 deg.C and is subjected to a centrifugal sepn. for 5min at 1,500rpm to take out only the lymph cells. Such lymph cells are cleaned with a physiological salt soln. and the lymph cells after cleaning which is held suspended in 300mul physiological salt soln. is introduced into an electron spin resonator (ESR device) and while UV rays are irradiated thereto, the quantitative measurement is made. The result measured in such a way indicates that the intensity (upper part in the figure) indicated by the cancer patient's lymph cells is about 4-times the intensity (lower part in the figure) indicated by the normal healthy person's lymph cells. The exact identification test is thus accomplished by using the hematoporphyrin deriv.

Description

【発明の詳細な説明】 本発明は、癌罹病を検定する方法に関し、特に生体外（
ｉｎ　ｖｉｔｒｏ）においてリンパ球と結びついたへマ
ドポルフィリン誘導体の岳を電子スピン共鳴により測定
することにより検定するようにした方法に関する。。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for assaying cancer morbidity, particularly in vitro (
The present invention relates to a method for assaying by measuring the amount of hemadoporphyrin derivatives bound to lymphocytes in vitro by electron spin resonance. .

急増しつつあるガンの診断法として最近脚光を浴びてき
たものの１つに光感受性物質とレーザ光線を用いた方法
がある。これは、ＩＩ！Ｉ！瘍に親和性を有した光感受
性物質を静脈内投与し、長時間かけＣ腫瘍に蓄積させた
後、レーザ光線を患部に照射し、例えばレーザ光照射叫
よって光感受性物質が発Ｊる蛍光を観察することによっ
て腫瘍を判別するものである。One method that has recently attracted attention as a diagnostic method for the rapidly increasing number of cancers is a method that uses a photosensitizer and a laser beam. This is II! I! A photosensitizer that has an affinity for tumors is intravenously administered, allowed to accumulate in the C tumor over a long period of time, and then a laser beam is irradiated to the affected area to emit fluorescence emitted by the photosensitizer. Tumors are identified by observation.

どころが、この方法は生体内（ｉｎ　ｖｉｖｏ　）での
測定であり、蓄積までに３日間程度必要であり、患者の
苦痛はもちろんのこと多くの手間がかかつていた。However, this method involves in vivo measurement and requires about 3 days for accumulation, which not only causes pain for the patient but also requires a lot of effort.

イこで、本発明者は、先にへマドポルフィリンが生体外
に取出した癌細胞やリンパ球によっても摂取されること
に看目し、生体外に取出した癌細胞やリンパ球にへマド
ポルフィリンを混入し、癌細胞やリンパ球と結びついた
へマトボルフイリンの量を測定することにより癌罹病を
検定する方法を特願昭５８−８３６５１号として出願し
た。本発明者は更にｒ１１１究を重ね、へマドポルフィ
リンよりもへマドポルフィリン誘導体を用いた方がさら
に良好な、検定結墨が得られることを見出した・従って
本発明は、へマドボルフ４リン誘尋体を用いることによ
り癌罹病を検定する方法を提供するもので、リンパ球に
へマドポルフィリン１６体を混入し、リンパ球と結びつ
いたへマドポルフィリン誘導体の量を電子スピン共鳴に
より定量測定づるようにしたことを特徴としている。以
下、図面を用いて本発明を詳述する。In this regard, the present inventor noticed that hemadoporphyrin is also ingested by cancer cells and lymphocytes taken out of the body, and the present inventors discovered that hemadoporphyrin was ingested by cancer cells and lymphocytes taken out of the body. Japanese Patent Application No. 58-83651 has been filed for a method for assaying cancer disease by mixing hematovolufilin with cancer cells and measuring the amount of hematovolufilin associated with cancer cells and lymphocytes. The present inventor conducted further research and found that even better test results could be obtained by using hemadoporphyrin derivatives than by hemadoporphyrin. Therefore, the present invention provides hemadorphyrin 4-phosphorus inducer. This method provides a method for assaying cancer morbidity using the human body, in which 16 hematoporphyrins are mixed into lymphocytes, and the amount of hematoporphyrin derivatives bound to lymphocytes is quantitatively measured by electron spin resonance. It is characterized by what it did. Hereinafter, the present invention will be explained in detail using the drawings.

へマドポルフィリン誘導体は、へマドポルフィリンと全
く同様に、光照射を受ｔプるとπ−カチオンラジカルを
生成し、このラジカルを電子スピン共鳴装置（Ｅ’ＳＲ
装置）で測定することかできることを本発明者は確認し
た。第１図は上記ラジカルのＥＳＲスペクトルの測定例
を示すもので、ｑ値は２．００１５である。第２図はへ
マドポルフィリン誘導体のリン酸バッファ溶液約１００
μ之に光（紫外線）を照射して測定したＥＳＲスペクト
ル強度（第１図におけるビーク−ピーク値ｈ）と希釈８
ｉＩ１度との関係を示しており、高濃度領域に飽和現象
が見られるものの、全体としてへマドポルフィリン誘導
体濃度とＥＳＲ信号強度との間には、へマドポルフィリ
ンと全く同様に良好な直線関係が存在することが分る。Just like hematoporphyrin, hematoporphyrin derivatives generate π-cation radicals when exposed to light, and these radicals are analyzed using an electron spin resonance apparatus (E'SR).
The present inventor has confirmed that the measurement can be performed using a device (device). FIG. 1 shows a measurement example of the ESR spectrum of the above radical, and the q value is 2.0015. Figure 2 shows a phosphate buffer solution of hematoporphyrin derivatives containing approximately 100%
ESR spectrum intensity (beak-peak value h in Figure 1) measured by irradiating light (ultraviolet light) to μ and dilution 8
Although a saturation phenomenon is observed in the high concentration region, overall there is a good linear relationship between the hematoporphyrin derivative concentration and the ESR signal intensity, just as with hematoporphyrin. I know it exists.

尚、測定は、室温では感度が低下するため、−１００℃
の低温で行われＣいる。In addition, the measurement should be performed at -100℃ because the sensitivity decreases at room temperature.
It is carried out at a low temperature of C.

第３図は、ＥＳＲ装置を用いて本発明にかかる方法を実
施する際の手順を示す流れ図であり、（１）患者から採
取した血液からリンパ球のみを例えば遠心分離によって
取出し、（２）これを例えば０．００５モルのへマドポ
ルフィリン誘導体“　５０μ込と混合し、（３）３７℃
で約１′０分間インキュベ゛−トシ、（４）　１５００
ｒｌ）Ｉｌｌ、で５分間遠心分離してリンパ球のみ取出
して生理食塩水にて洗浄し、（５）洗浄後リンパ球を３
００μ込の生理食塩水に浮遊させた状態でＥＳＲ装置に
導入し、光（紫外線）を照射しつつ定量測定を行う。FIG. 3 is a flowchart showing the procedure for carrying out the method according to the present invention using an ESR device, in which (1) only lymphocytes are removed from blood collected from a patient by, for example, centrifugation; (3) 37°C
Incubate for approximately 1'0 minutes at (4) 1500 ml.
(rl) Ill, centrifuged for 5 minutes to remove lymphocytes and wash with physiological saline. (5) After washing, the lymphocytes were
The sample is introduced into an ESR device while suspended in physiological saline containing 00μ, and quantitative measurements are performed while irradiating it with light (ultraviolet light).

第４図は、第３図の手順に従って測定した正常健康者（
下）と癌患者（上）のリンパ球についてのＥＳＲスペク
トルを大々示す。癌患者は、正常健康者に対し約４４８
の強度になっていることが分る。Figure 4 shows normal healthy subjects (
The ESR spectra of lymphocytes from a cancer patient (bottom) and a cancer patient (top) are shown in detail. Cancer patients are approximately 448 times older than normal healthy people.
It can be seen that the strength is as follows.

第５図は、正常健ル者２１名、癌患者２６名、膠原病患
者５名から採取した血液について、第３図の手順で測定
した測定結果を示す。各測定結果は、測定時に混入した
一定ωの標準試ｒ１による基準ピーク（第４図における
Ｒ′）のピーク−ピーク値ｈｒとの比のかたちで相対強
度１泊として表わしている。第５図から、癌患者は相対
強度が０．８以上を示１のに対し、癌ではない正常１１
ｔ［者及びＩｌｌ原病患者′はそれ以下の値を示し、あ
るレベルを、境界線として癌にかかつているか否かを検
定することが可能であることが分る。しかｂｌへマドポ
ルフィリンを用いる特願昭５８−８３６５１号の方法で
は、境界線付近で多少癌患者と正常健康者の値がオーバ
ーラツプする部分が□存在したが、本発明ではそのよう
なオーバーラツプがなくなり、癌にかかっているか否か
を更に明確に判別することが可能となった。FIG. 5 shows the measurement results of blood collected from 21 normal healthy subjects, 26 cancer patients, and 5 collagen disease patients using the procedure shown in FIG. 3. Each measurement result is expressed as a relative intensity per night in the form of a ratio to the peak-to-peak value hr of the reference peak (R' in FIG. 4) obtained by the standard test r1 with a constant ω mixed in during the measurement. From Figure 5, cancer patients show a relative intensity of 0.8 or higher1, while non-cancer normals show a relative intensity of 11.
It can be seen that t[ and Ill disease patients' show values lower than this, and it is possible to use a certain level as a borderline to test whether or not they have cancer. However, in the method of Japanese Patent Application No. 1983-83651 using bl hemadoporphyrin, there was a portion where the values of cancer patients and normal healthy people overlapped somewhat near the border line, but in the present invention, such overlap is eliminated. It has now become possible to more clearly determine whether or not someone has cancer.

尚、混入する標準試料の量ににって基準ピークの強度が
変わるので、相対強度値も変わる。従って、正常健康者
と癌患者との相対強度値での比較は、同じ量の標準試料
を混入し−（行わなければならないことは言うまでもな
い。Note that since the intensity of the reference peak changes depending on the amount of the standard sample mixed in, the relative intensity value also changes. Therefore, it goes without saying that a comparison of relative intensity values between a normal healthy person and a cancer patient must be performed by mixing the same amount of standard sample.

又、上述した実施例ではへマドポルフィリン誘導体をリ
ンパ球に混入した後に光を照射し、ラジカルを生成させ
てＥＳＲ測定したが、ラジカルをイｊする標識物質を予
め結合させることにより標識化したへマドポルフィリン
誘導体を用いることも可能であり、そうすればへマドポ
ルフィリン誘導体が始めからラジカルを有するので、光
を照射しなくてもＥ’ＳＲ装置によりリンパ球と結びつ
いたヘマトポルフィリン誘導体の量を測定することが口
Ｊ能である。　′　□ 以上詳述した如く、本発明によればへマドポルフィリン
誘導体を用いることによりへマドポルフィリン誘導体を
用いた場合よりも更に正確にｗＩ罹病を検定することの
できる方法が実現される。In addition, in the above-mentioned example, a hematoporphyrin derivative was mixed into lymphocytes and then irradiated with light to generate radicals for ESR measurement. It is also possible to use a hematoporphyrin derivative, and since the hematoporphyrin derivative has radicals from the beginning, it is possible to measure the amount of hematoporphyrin derivative bound to lymphocytes using an E'SR device without irradiating it with light. To do so is Kuchijō. ' □ As described in detail above, according to the present invention, by using a hemadoporphyrin derivative, a method that can more accurately assay wI disease than in the case of using a hemadoporphyrin derivative is realized.

【図面の簡単な説明】[Brief explanation of drawings]

第１図はへマドポルフィリン誘導体のＥＳＲスペクトル
の測定例を示す図、第２図はへマドポルフィリン１１体
ＩＩＩとＥＳＲスペクトル相対強痕との関係を示す図、
第３図はＥＳＲ装置を用いて本発明にかかる方法を実施
する際の手順を示す流れ図、第４図は第３図の手順に従
って測定した正常健康者（下）と癌患者（上）のリンパ
球についてのＥＳＲスペクトルを示す図、第５図は、正
常健康者２１名、癌患者２６名、膠原病患者５名から採
取した血液について第３図の手順で測定した測定結果を
示す図である。 手続補正書く自発） 昭和５９年２月　１日 １、事件の表示 昭和５８年特許願第２４３４５３号 ２、発明の名称 癌罹病を検定Ｊ゛る方法 ３、補正をする者 事件との関係　特許出願人 住所　東京都昭島市中神町１４１８番地（Ｔ　Ｅ　Ｌ　
０４２５　（４３）　１１６５）４、補正の対象 発明の詳細な説明の欄 ５、補正の内容 明ＩＩ書第６頁２０行目を次の通り補正する。 「イリンを用いた場合よりも更に正確に癌罹」以上 手続柑ｌ正訓　（自発） １．事件の表示′ 昭和５８年特許願第２４３４５３号 ２、発明の名称 癌罹病を検定Ｊる方法 ３、補正をする者 事件との関係　特許出願人 住所　東京都昭島市中神町１４１８番地（Ｔ　Ｅ　Ｌ　
０４２５　（４３）　１１６５）４、補正の対象 明１ｌｌｌ書全文 ５、補正の内容 （１）明細書全文を別紙の通り補正する。 以　上 明　細　書 １、発明の名称 リンパ球にｐＡＪる鑑別テスト　□ ２、特許請求の範囲　′　□ ３、発明の詳細な説明 ［産業上の利用分野］ 本発明はリンパ球に関するｇ沖１テストに関し、特に癌
の診断に用いて好適な鑑別テストに関する。 ［従来の技術］ 急増しつつあるガンの診断法として最近脚光を浴びてき
たものの１つに光感受性物質とレーザ光線を用いた方法
がある。これは、腫瘍に親和性を有した光感受性物質を
静脈内投与し、長時・間かけて腫瘍に蓄積させた後、レ
ーザ光線を患部に照射し□、例えばレーザ光照射によっ
て光感受性物質が発する蛍光を観察することによって腫
瘍を判別するものである。 ［発明が解決しようどする問題点］ ところが、この方法は生体内（ｉｎ　ｖｉｖｏ　）での
測定であり、蓄積までに３日問程度必要であり、患者の
苦痛はもちる／υのこと多く６手問がかかっていた。 そこで、本発明者は、先にへマドポルフィリンが生体外
に取出したリンパ球によって摂取されることに着目し、
生体□外に取出したリンパ球にへマドポルフィリンを添
加し、リンパ球に含まれるラジカルの缶を電子スピン共
鳴装置を用いて測定する鑑別テストを特願昭５８’＝８
”３６５１号として出願した。本発明者は更に研究を重
ね□、へマドポルフィリンよりもへやトボルフィリン誘
導体を用いた方がさらに良好な検定結果が得られること
を見出した。 ［問題点を解決するだめの構成］ 従って本発明は、へマドポルフィリン誘導体を用いるリ
ンパ球に関する鑑別テストを提供するもので、被験者か
らのリンパ球にへマドポルフィリン誘導体を添加し、イ
ンキュベート後リンパ球のみを分離し、分離したリンパ
球に含まれるラジカル量を電子スピン共鳴装置を用いて
定６測定する、諸工程より成ることを特徴としている。 以下、図面を用いて本発明を詳述する。 ［実施例］ へマドポルフィリン誘導体は、へマドポルフィリンと全
く同様に、光照射を受けるとπ−カチオンラジカルを生
成し、このラジカルを電子スピン共鳴装置（ＥＳＲ装置
）で測定することができることを本発明者は確認した。 第１図は上記ラジカルのＥＳＲスペクトルの測定例を示
づもので、Ｑ値は２．００１５である。第２図はへマド
ポルフィリン誘導体のリン酸バッファ溶液約１００μ込
に光（紫外ＷＡ）を照射し゛Ｃ測定したＥＳＲスペクト
ル強度（第１図におけるピーク−ピーク値ｒ〕）と希釈
濃度との関係を示しており、高濃度領域に飽和現象が見
られる一ｂのの、全体としてへマドポルフィリン誘導体
濃度とＥＳＲ信号強度との間には、へマドポルフィリン
と全く同様に良好な直線関係が存在Ｊ”ることが分る。 尚、測定は、室温では感度が低下するため、−１００’
Ｃの低温で行われている。 第３図は、ＥＳＲ８置を用いて本発明にかかる方法を実
施する際の手順を示す流れ図であり、（１）患者から採
取した血液からリンパ球のみを例えば遠心分離によって
取出し、く２）これを例えば０．００５モルのへマドポ
ルフィリンＹｍ体５０μ込と混合し、（３）３７℃で約
１０分間インキュベートし、（４）　１５００ｒｐｍ、
で５分間遠心分離してリンパ球のみ取出して生理食塩水
にて洗浄し、（５）洗浄後リンパ球を３００μ之の生理
食塩水に浮遊させた状態でＥＳＲ装置に導入し、光（紫
外線）を照射しつつ定ｍ測定を行う。 第４図は、第３図の手順に従って測定した正常健康者（
下）と癌患者（上）のリンパ球についてのＥＳＲスペク
トルを夫々承り。癌患者のリンパ球が示ず強度は、正常
健泉者のリンパ球が示す強度の約４倍になっていること
が分る。 第５図は、正常健康者２１名、癌患者２６名、膠原病患
者５名から採取した血液について、第３図の手順で測定
した測定結果を示す。各測定結果は、測定時に混入した
一定ωの標準試料による基準ピーク（第４図におけるＲ
）のピーク−ピークＩｔ１ｈｒとの比のかたちで相対強
度値として表わしている。第５図から、−癌患者のリン
パ球は相対強度が０．８以上を示すのに対し、癌ではな
い正常健康者及び膠原病患者のリンパ球はそれ以下の値
を示す。従って、本発明の鑑別テストによれば、あるレ
ベルを境界線として癌患者のリンパ球か否かを検定する
ことが可能であることが分る。しかも、へマドポルフィ
リンを用いる特願昭５８−８３６５１号の方法では、境
界線付近で多少癌患者と正常を座者の値がオーバーラツ
プする部分が存イｔしたが、本発明ではそのようなオー
バーラツプがなくなり、癌にかかっているか否かを更に
明確に判別することが可能となった。 尚、混入する標準試料の聞によって基準ピークの強度が
変わるので、相対強度値も変わる。従って、正常健康者
と癌患者との相対強度値での比較は、同じ量の標準試料
を混入して行わなければならないことは言うまでもない
。 又、上述した実施例ではへマドポルフィリン誘導体をリ
ンパ球に混入した後に光を照射し、ラジカルを生成さけ
てＥＳＲ測定したが、ラジカルを有する標識物質を予め
結合さぼることにより標識化したへマドポルフィリン誘
導体を用いることも可能であり、そうづればへマドポル
フィリン誘導体が始めからラジカルを有づるので、光を
照射しなくてもＥＳＲ装置によりラジカル量を測定する
ことが可能中ある。 ［効果］ 以上詳述した如く、本発明によればへマドポルフィリン
誘導体を用いることによりへマｉ・ポルフィリンを用い
る場合よりも更に正確な鑑別テスト　−が実現される。 ４、図面の簡単な説明 第１図はへマドポルフィリン誘導体のＥＳＲスペクトル
の測定例を示す図、第２図はへマドポルフィリン誘導体
濃度とＥＳＲスペクトル相対強度どの関係を示す図、第
３図はＥＳＲ装置を用いて本発明にかかる方法を実施す
る際の手゛順を示す流れ図、第４図は第３図の手順に従
って測定した正常蛯座者（下）と癌患者（上）のリンパ
球についてのＥＳＲスペクトルを示１図、第５図は、正
常Ｒ東名２１名、癌患者２６名、膠原病患者５名から採
取した血液について第３図の手順で測定した測定結果を
示す図である。FIG. 1 is a diagram showing a measurement example of the ESR spectrum of a hemadoporphyrin derivative, FIG. 2 is a diagram showing the relationship between hemadoporphyrin 11 substance III and the relative intensity of the ESR spectrum,
Figure 3 is a flowchart showing the procedure for carrying out the method according to the present invention using an ESR device, and Figure 4 shows the lymph nodes of a normal healthy person (bottom) and a cancer patient (top) measured according to the procedure in Figure 3. Figure 5 is a diagram showing the ESR spectrum of the sphere, and is a diagram showing the measurement results of blood collected from 21 normal healthy people, 26 cancer patients, and 5 collagen disease patients using the procedure shown in Figure 3. . (Spontaneous writing of procedural amendments) February 1, 1980 1, Indication of the case 1988 Patent Application No. 243453 2, Name of the invention Method for testing cancer disease 3, Person making the amendment Relationship with the case Patent application Address: 1418 Nakagami-cho, Akishima City, Tokyo (TEL
0425 (43) 1165) 4. Column 5 of the detailed description of the invention subject to amendment, page 6, line 20 of the Statement of Contents of Amendment II is amended as follows. ``Cancer infection is more accurate than when using Ilin.'' The above procedure is a true lesson (voluntary) 1. Indication of the case Patent Application No. 243453 of 1983 2, Title of the invention Method for testing cancer morbidity 3, Person making the amendment Relationship to the case Patent applicant address 1418 Nakagami-cho, Akishima-shi, Tokyo (TEL
0425 (43) 1165) 4. The entire text of the document to be amended 5. Contents of the amendment (1) The entire text of the specification will be amended as shown in the attached sheet. Specification 1. Name of the invention: Differential test for pAJ on lymphocytes □ 2. Scope of claims ' □ 3. Detailed description of the invention [Field of industrial application] The present invention is a test for differential pAJ on lymphocytes. In particular, the present invention relates to a differential test suitable for use in cancer diagnosis. [Prior Art] One method that has recently attracted attention as a diagnostic method for cancer, which is rapidly increasing in number, is a method using a photosensitizer and a laser beam. This involves intravenously administering a photosensitizer that has an affinity for tumors, allowing it to accumulate in the tumor over a long period of time, and then irradiating the affected area with a laser beam. Tumors are identified by observing the fluorescence they emit. [Problems to be solved by the invention] However, this method is an in vivo measurement, requires about 3 days to accumulate, and is often painful to the patient.6 I was being questioned. Therefore, the present inventor focused on the fact that hemadoporphyrin is ingested by lymphocytes taken out of the body,
A patent application was filed in 1982 for a differential test in which hematoporphyrin was added to lymphocytes taken out of the living body and the amount of radicals contained in the lymphocytes was measured using an electron spin resonance device.
The present inventor conducted further research and found that even better assay results could be obtained by using heyatovorphyrin derivatives than hematoporphyrin. [Problems solved. Composition of Sudame] Therefore, the present invention provides a differential test for lymphocytes using a hemadoporphyrin derivative, in which the hemadoporphyrin derivative is added to lymphocytes from a subject, and after incubation, only the lymphocytes are separated. It is characterized by consisting of various steps of constantly measuring the amount of radicals contained in separated lymphocytes using an electron spin resonance apparatus.The present invention will be described in detail below with reference to the drawings.Go to [Example] The present inventors have confirmed that, just like hemadoporphyrin, madoporphyrin derivatives generate π-cation radicals when irradiated with light, and these radicals can be measured with an electron spin resonance device (ESR device). Figure 1 shows an example of measuring the ESR spectrum of the above radical, and the Q value is 2.0015. Figure 2 shows an example of measuring the ESR spectrum of the above radical, with a Q value of 2.0015. It shows the relationship between the ESR spectrum intensity (peak-to-peak value r in Figure 1) measured by irradiation with C and the dilution concentration. It can be seen that a good linear relationship exists between the porphyrin derivative concentration and the ESR signal intensity, just as in the case of hematoporphyrin. In addition, since the sensitivity decreases at room temperature, the measurement is performed at -100'
It is carried out at a low temperature of C. FIG. 3 is a flowchart showing the procedure for carrying out the method according to the present invention using the ESR8 device, in which (1) only lymphocytes are removed from blood collected from a patient by, for example, centrifugation, and (2) this is carried out. For example, 0.005 mol of hematoporphyrin Ym is mixed with 50 µ of hematoporphyrin Ym, (3) incubated at 37°C for about 10 minutes, (4) 1500 rpm,
(5) After washing, the lymphocytes were suspended in 300 μm of physiological saline and introduced into an ESR device, and exposed to light (ultraviolet light). Perform constant m measurement while irradiating. Figure 4 shows normal healthy subjects (
We received ESR spectra of lymphocytes from a cancer patient (bottom) and a cancer patient (top). It can be seen that the intensity exhibited by the lymphocytes of cancer patients is about four times that of the lymphocytes of normal healthy individuals. FIG. 5 shows the measurement results of blood collected from 21 normal healthy subjects, 26 cancer patients, and 5 patients with collagen disease using the procedure shown in FIG. Each measurement result is based on the reference peak (R
) is expressed as a relative intensity value in the form of a ratio of peak to peak It1hr. From FIG. 5, - Lymphocytes of cancer patients show a relative intensity of 0.8 or more, whereas lymphocytes of normal healthy people without cancer and patients with collagen disease show values lower than that. Therefore, it can be seen that according to the differential test of the present invention, it is possible to test whether lymphocytes are cancer patient lymphocytes or not, using a certain level as a borderline. Moreover, in the method of Japanese Patent Application No. 83,651/1983 using hematoporphyrin, there was a portion where the values of cancer patients and normal sitters overlapped to some extent near the borderline, but the present invention eliminates such overlap. This has made it possible to more clearly determine whether a patient has cancer or not. Note that since the intensity of the reference peak changes depending on the amount of the standard sample mixed in, the relative intensity value also changes. Therefore, it goes without saying that a comparison of relative intensity values between a normal healthy person and a cancer patient must be performed by mixing the same amount of standard sample. In addition, in the above-mentioned example, a hemadoporphyrin derivative was mixed into lymphocytes and then irradiated with light to avoid generation of radicals and perform ESR measurement. It is also possible to use a derivative; in that case, since the hemadoporphyrin derivative has radicals from the beginning, it is possible to measure the amount of radicals with an ESR device without irradiating it with light. [Effects] As detailed above, according to the present invention, by using a hematoporphyrin derivative, a more accurate discrimination test is realized than when using hematoporphyrin. 4. Brief explanation of the drawings Figure 1 is a diagram showing a measurement example of the ESR spectrum of a hematoporphyrin derivative, Figure 2 is a diagram showing the relationship between the hematoporphyrin derivative concentration and the relative intensity of the ESR spectrum, and Figure 3 is a diagram showing the ESR spectrum. A flowchart showing the steps for carrying out the method according to the present invention using the device. Figure 4 shows the lymphocytes of a normal Scorpio subject (bottom) and a cancer patient (top) measured according to the procedure of Figure 3. Figures 1 and 5 show the ESR spectra of blood collected from 21 normal R Tomei patients, 26 cancer patients, and 5 collagen disease patients, and the results were measured using the procedure shown in Figure 3.

Claims (3)

【特許請求の範囲】[Claims] （１）リンパ球にへマドポルフィリン誘導体を混入し、
リンパ球と結びついたへマドポルフィリン誘導体の量を
電子スピン共鳴により定量測定するようにした癌罹病を
検定づる方法。(1) Mixing hemadoporphyrin derivatives into lymphocytes,
A method for assaying cancer morbidity in which the amount of hemadoporphyrin derivatives bound to lymphocytes is quantitatively measured by electron spin resonance. （２）混入後へマドポルフィリン誘導体に光を照射し、
光照射により生成されたラジカルを標識として電子スピ
ン共鳴により定量測定が行われる特許請求の範囲第１項
記載の検定方法。(2) Irradiate the hemadoporphyrin derivative with light after mixing,
2. The assay method according to claim 1, wherein quantitative measurement is performed by electron spin resonance using radicals generated by light irradiation as labels. （３）へマドポルフィリン誘導体は予めラジカルを右す
る物質によって標識化されＣ１１５す、そのラジカルを
標識として電子スピン共鳴により定ｍ測定が行われる特
許請求の範囲第１項記載の検定方法。(3) The assay method according to claim 1, wherein the hemadoporphyrin derivative is previously labeled with a substance that acts as a radical, and the constant measurement is performed by electron spin resonance using the radical as a label.
JP24345383A 1983-05-13 1983-12-23 Method for inspecting contraction of cancer Granted JPS60135765A (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP24345383A JPS60135765A (en) 1983-12-23 1983-12-23 Method for inspecting contraction of cancer
US06/608,996 US4696905A (en) 1983-05-13 1984-05-10 Method for diagnosing cancer using ESR
DE8484105337T DE3483493D1 (en) 1983-05-13 1984-05-11 METHOD FOR DETECTING CANCER CELLS.
DE198484105337T DE125651T1 (en) 1983-05-13 1984-05-11 METHOD FOR DETECTING CANCER CELLS.
EP84105337A EP0125651B1 (en) 1983-05-13 1984-05-11 A method for detecting cancerous cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP24345383A JPS60135765A (en) 1983-12-23 1983-12-23 Method for inspecting contraction of cancer

Publications (2)

Publication Number Publication Date
JPS60135765A true JPS60135765A (en) 1985-07-19
JPH035551B2 JPH035551B2 (en) 1991-01-25

Family

ID=17104109

Family Applications (1)

Application Number Title Priority Date Filing Date
JP24345383A Granted JPS60135765A (en) 1983-05-13 1983-12-23 Method for inspecting contraction of cancer

Country Status (1)

Country Link
JP (1) JPS60135765A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62182663A (en) * 1985-10-23 1987-08-11 Nippon Mejifuijitsukusu Kk Radioactive metal-labelled cancer diagnostic agent
JP2010054305A (en) * 2008-08-27 2010-03-11 Tohoku Denshi Sangyo Kk Method for acquiring data used for cancer contraction verification

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59208460A (en) * 1983-05-13 1984-11-26 Jeol Ltd Method for measuring bonding degree of cell and hematoporphyrin
JPS6025452A (en) * 1983-07-23 1985-02-08 Supeshiaru Refuarensu Lab:Kk Inspection of cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59208460A (en) * 1983-05-13 1984-11-26 Jeol Ltd Method for measuring bonding degree of cell and hematoporphyrin
JPS6025452A (en) * 1983-07-23 1985-02-08 Supeshiaru Refuarensu Lab:Kk Inspection of cancer

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62182663A (en) * 1985-10-23 1987-08-11 Nippon Mejifuijitsukusu Kk Radioactive metal-labelled cancer diagnostic agent
JPH0424661B2 (en) * 1985-10-23 1992-04-27 Nippon Mejifuijitsukusu Kk
JP2010054305A (en) * 2008-08-27 2010-03-11 Tohoku Denshi Sangyo Kk Method for acquiring data used for cancer contraction verification

Also Published As

Publication number Publication date
JPH035551B2 (en) 1991-01-25

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