JP3655741B2 - Immune capacity measuring method and its evaluation system - Google Patents

Description

【００２２】
次に第２ステップとして、標的細胞(ターゲットセル,ＴＣ)としての一般的な免疫能測定用細胞Ｋ５６２標識プロトコルについて説明する。細胞調整はデキストラン硫酸を1.5mlの遠心管に５mg計量し、ここではＫ５６２が５×10⁶個になるように加え、４℃で遠心分離し、上澄を除去し、バッファーＡの置換操作を２回繰返した後、Ｅｕ−ＤＴＰＡによるラベリングバッファを調整した。この操作は標準ステップでなされる。標識物質についてはユーロピウムのかわりに放射性クロム51を用いた標識で代替できる。この際の方法は標的細胞にクロムを加え37℃のＣＯ₂インキュベーター中で１時間培養すればよい。またＫ５６２に代えて、被検者個人のガン細胞を取り出して標識をすることができ、この場合は各個人に投与すべき免疫能の賦活物質を的確に選定することが可能となる。
【００２３】
作成したラベリングバッファーを複数のチューブに0.5mlずつ入れ、氷冷してこれにバッファーＢを加え撹拌、次いで遠心分離と上清の除去を２回繰返した。この操作により、細胞内を安定化させて標的細胞(ＴＣ)とした。
【００２４】
標的細胞は、更にＲＰＭＩを２mlずつ加えてピペッティングして撹拌し、その後４℃での遠心分離と上清の除去を上記と同様に３回繰り返し、１〜２時間放置冷蔵したもののガン細胞Ｋ５６２の細胞数をカウントして５×10⁴／mlになるようにＲＰＭＩで希釈して標的細胞液とした。そして、この溶液に対して表１の下段に示す抗ガン剤のマイトマイシンＣをガン細胞傷害物質として添加前処理して標的ガン細胞処理液とした。
【００２５】
細胞傷害活性試験液は、この標的細胞液と上述の第１ステップで述べたリンパ球培養原液と所定比率で混合して調整し、リンパ球の免疫能の測定に供した。そのために、ガン細胞：リンパ球の比率が常に同じになるように細胞数を調整した。例えば、リンパ球1,000個に対し細胞数200個の５：１から10：１，20：１，40：１などの希釈系列の試料につき試験をした。
【００２６】
免疫能は前述した公知の細胞傷害活性試験により測定することができるが、ここでは、放射性同位元素⁵¹Ｃｒによる放出試験(Ｃｒ法)か、あるいは更に進んでユーロピウム元素による放出試験(Ｅｕ法)が最もふさわしい。具体的な検査法は、Ｃｒ法についてはラジオイムノアッセイ、Ｅｕ法についてはフルオロイムノアッセイとして確立されている方法に準拠した。
【００２７】
まず、放射性同位元素⁵¹Ｃｒを用いたガン細胞の標識を行う。それを標的細胞(ＴＣ)として適当な比率でエフェクターであるリンパ球を混ぜ、それに標的細胞が傷害されたときに上澄に出てくる⁵¹Ｃｒをガンマカウンターで測定するというものである。この方法は放射性物質を用いることから、特別な施設を必要としたり、放射性廃棄物処理の問題や放射線を発する⁵¹Ｃｒによる被爆の問題があり、この方法と相関性の優れるユーロピウム(Ｅｕ)−ＤＴＰＡキレート法で行う方がより簡便である。これをユーロピウム放出試験という。ユーロピウム放出試験は、ガン細胞をＥｕ−ＤＴＰＡキレートで標識した標的細胞を用い、ＮＫ(Natural Killer)およびＬＡＫ(Lymphokine Activatid Killer)細胞により、ＭＨＣ非拘束性Ｔ細胞またはＮＫＴ細胞により標的細胞を破壊し、遊離したＥｕ−ＤＴＰＡ量を時間分解蛍光法により測定するのである。放出試験の具体的方法は公知であるので、詳細は省略するが、本発明には特に、ユーロピウム放出試験が最もふさわしい方法である。
【００２８】
以上のように、ガン細胞を放射性向位元素⁵¹Ｃｒでの標的細胞(ＴＣ)とし、適当な比率でエフェクターであるリンパ球を混ぜてそれによって標的細胞が傷害されたときに上清に出てくる⁵¹Ｃｒをガンマカウンターで測定するか、あるいは、Ｅｕ−ＤＴＰＡで標識した標的細胞をエフェクターのリンパ球と混ぜて、上清に出てくるＥｕを蛍光光度計で測定するが、これらの自動測定システムのブロック図を図１に示す。各ユーロピウム(Ｅｕ)放出試験サンプルは、まず検体名登録をして記録し、プレートデータ作成した後、蛍光光度計で測定して測定データを収集、記録するのである。
【００２９】
すなわち、測定データのコンピュータ処理は次の順序で行うのである。蛍光光度計には、商品名デルフィア1234蛍光光度計または、アルボ1420蛍光光度計が計測したデータをCCITT V24インターフェースを介してコンピュータへ送信し、コンピュータの一時記憶装置に測定データを蓄積する。そのデータに対して予め検体名を登録し、特定の番号を割付けてそのデータと共に大容量記憶装置に蓄積する。大容量記憶装置に蓄積した測定データを一時記憶装置に呼び出して、測定グラフデータとし、これらを印字形式に編集し、％細胞傷害活性を算出する。
【００３０】
ここでいう検体名登録とは、賦活物質の具体名のディスク装置への登録であり、例えばサイトカインのインターロイキンＩＬ２、漢方生薬の場合、小柴胡湯,補中益気湯などの物質名の登録をする。蛍光光度計測定容器のどの位置に上記検体のどれが入っているかを明確にしたものがプレートデータであり、これも上記同様ディスク装置へ登録し、プレートデータリストを確認する。
【００３１】
次いで、測定は蛍光光度計によるが、ここではワラック社(WALLAC)のデルフィア1234蛍光光度計を用い、Ｅｕの個数を測定し、そのデータを収集する。すなわち、標的細胞がＥｕ−ＤＴＰＡでどの程度ラベルされたかを「spontaneous Eu3+ release」(Ｓ)と「maximum Eu3+ release」(Ｍ)の比Ｓ／Ｍの百分率を％細胞傷害活性として表示したデータを作成し、グラフ化するのである。
【００３２】
Ｅｕ−ＤＴＰＡによる標的細胞を用いる場合、ファルマシアバイオテクノロジー社の「デルフィアＥｕ−ラベリングキット」での測定のフローチャートを図２に示す。すなわち、測定データの編集画面は、大きく「測定データ収集画面」、「測定データリスト表示画面」、「プレートデータ作成画面」、「プレートデータリスト表示画面」及び「検体名登録画面」に分けられる。更に、これらの画面はそれぞれ▲１▼〜▲５▼の作業項目へと移行する。
【００３３】
図２は測定データの基本作成フローチャートであり、測定作業を行うための基本機能を表している。ここでは作業項目は単に▲１▼〜▲５▼で表示されているが、測定データ収録画面の作業項目▲１▼が主流であり、▲２▼〜▲５▼は▲１▼の補助的機能であるので省略し、▲１▼について更に説明を加える。
【００３４】
図３は図２の▲１▼からの続行フローであって、蛍光光度計デルフィアからのデータ収集フローチャートである。ここで測定データ保存画面をみながら被検者(患者)番号,氏名,プレートデータを入力し、測定データファイルを作成し、表計算ファイルとし、表計算プログラムを作動させて次のフローへ移る。
【００３５】
図４は測定データから％細胞傷害活性算出のフローチャートであり、図３中の▲６▼から続行して行う表計算のための内部処理を表したものである。図５は測定印刷フォーマット作成フローチャートであり、図４の▲９▼からの続行作業項目を示す。ここでは、印字フォーマットを確定し、グラフとデータの表を印字、プリントアウトするフローを表している。
【００３６】
ここで先に述べた図１の各種賦活物質の％細胞傷害活性に及ぼす影響につき得られた測定結果について具体的に説明する。表２は既述したリンパ球1,000個に対し、細胞数200個の５：１，10：１，20：１及び40：１の比率に希釈した採血時免疫能を％細胞傷害活性で示したもので、その値は10.4〜34.1を示した。
【００３７】
【表２】

【００３８】
表３は表１中のサイトカインとしてインターロイキン２(ＩＬ２)をリンパ球に添加し、またＢＲＭとしてのＯＫ４３２(ビシバニール)をリンパ球に添加しその％細胞傷害活性を調べたもので、これらの値は53.8〜96.3％と42.4〜84.4％という高い数値を得た。
【００３９】
【表３】

【００４０】
採取した血液中のリンパ球が持つガン細胞に対する攻撃力が傷害活性値で表されており、比率はリンパ球とガン細胞の数の関係が、40：１の場合はガン細胞１個に対してリンパ球が40個で攻撃する場合を示している。したがって、表２と表３の結果を対比すると、採血時免疫能に対してインターロイキン２を用いると約2.8〜5.2倍程度、ＯＫ４３２で約2.5〜4.1倍程度に増強できることを示していることがわかる。
【００４１】
表４はサイトカインと同様にして、漢方生薬の三陰三陽の主要処方のうちから、小柴胡湯等の５種につき、本発明の方法により％細胞傷害活性を求めたものを表している。この結果からいえることは、補中益気湯を被検者が服用すれば、採血時免疫能の約2.2〜2.9倍程度に服用後免疫能を増強できることと、その他４種の服用では免疫能を活性化し得ないことを示しており、これらの賦活物質についてのガン細胞に対するリンパ球の攻撃力評価に役立つことがわかる。
【００４２】
【表４】

【００４３】
表５は賦活物質として食品に含まれる有用物質のキトサンとアガリクス,微量元素としてのアルミニウムと、細胞傷害物質としての抗ガン剤(マイトマイシンＣ)を用いた場合の測定結果である。
【００４４】
【表５】

【００４５】
表５の結果から、この被検者においては、キトサンを服用すると採血時免疫能に比べて約10％程度免疫能が上昇するが、アガリクスでは約５％未満しか上昇していなく、このことによりキトサンとアガリクスではキトサンを服用する方が効率がよいことがわかる。加えて現在アルミニウムの弊害が叫ばれて久しいが、採血時免疫能に比べ80％程度に免疫能が低下することが予想されるため、この被検者は免疫能を上昇させる必要があるならば、なるべくアルミニウム製のものは使用しない方がよいといえる。次に、抗ガン剤であるマイトマイシンＣを生体に投与した場合であるが、抗ガン剤はそれ自身のガン細胞に対する傷害能力に加え、患者体内においてガン細胞を患者の免疫系に対して効率よく傷害させる能力も認められる。すなわち、この場合患者のリンパ球は何も投与されない場合より約２倍程度ガン細胞を傷害できることが予想され、この抗ガン剤の投与は患者にとって有効であることがわかる。また、ビタミンＣに関しても制ガン効果が報告されているが、患者がビタミンＣを服用すれば免疫能は1.5倍程度に上昇することが予想され、患者にとって利益があることを示している。
【００４６】
以上の表２〜５に示した数値データをもとにグラフ化し、各賦活物質の効果をわかりやすくしたのが、図６〜図８である。図６はインターロイキン２,ピシバニール及び５種類の漢方生薬の％細胞傷害活性を示す棒グラフであり、この被検者の血液免疫能活性化に対してインターロイキン２,ピシバニールおよび補中益気湯が極めて有効であることがわかる。その他ほとんど効果がないか、マイナスの効果を示すものも判定可能である。図７は被検者の免疫能がかなり高い値であるため、図６の被検者のような大きな上昇は期待できないが、キトサン,アガリクスにおいては若干の上昇が見られ服用しても免疫系に不利益になることはないが、アルミニウムではかなりの低下が見られ、使用しない方がよいということがわかる。図８は被検者の血液免疫能が21.7と低い、進行ガンの患者であるが、マイトマイシンＣの投与とビタミンＣの服用が生体のガン排除に有用な手段であることを示す。
【００４７】
【発明の効果】
以上のように、本発明の免疫能測定方法及びその評価システムによれば、従来、単にラジオイムノアッセイやフルオロイムノアッセイなどの免疫能測定方法はヒトの血液の免疫能測定にのみ用いられており、各種免疫能の賦活物質や逆に細胞傷害物質の効果につき定量的な値を求めることに応用されていなかったが、これらに薬効の定量化が可能となった。その結果、被検者個々に食養生や薬剤投与の的確な指導ができ、数値の定量化と継続的な測定により、これらの治療効果が判断できることとなった。
【００４８】
本発明では、上記測定方法をコンピュータ管理できるシステム化を完成したことで、血液リンパ球の賦活物質や抗ガン物質などの迅速、確実な評価が可能となった。
【００４９】
特に、ガン患者の治療に効果を発揮すると思われるが、ガン患者のみならず、健康体の人の免疫力向上を図る一般的な免疫力測定検査にも活用可能となる。すなわち、被検者自身の持っているリンパ球を活性化して免疫を上げる対策は賦活物質によるが、この方法は本来、食品あるいは食品中の有効成分によるのであるから、抗ガン剤のような薬剤に比べて副作用の心配がほどんどない。極めて安全な治療法である。本発明は、この点の応用を病院や保健センターなどの機関で広範囲に進めうる点で、極めて有用な免疫能測定方法及びその評価システムである。
【図面の簡単な説明】
【図１】免疫能自動測定システムのブロック図である。
【図２】測定データの基本作成フローチャートである。
【図３】測定デルフィアからのデータ収集フローチャートである。
【図４】測定データから％細胞傷害活性算出のフローチャートである。
【図５】測定印刷フォーマット作成フローチャートである。
【図６】各賦活物質による免疫能の変化を示すグラフである。
【図７】各賦活物質による免疫能の変化を示すグラフである。
【図８】細胞傷害物質及びビタミンＣによる免疫能の変化を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel method for measuring immunological capacity and an evaluation system therefor. The performance evaluation of an activator or anticancer substance for the immune capacity of human blood lymphocytes is performed quickly, accurately and efficiently, and the evaluation is performed by computer processing. By doing so, automation is possible.
[0002]
[Prior art]
As a method for measuring human immunocompetence, target cells as radioimmunoassay are labeled with ⁵¹ Cr, a radioactive substance, and then NK cells (Natural Killer) or LAK cells (Lymphokine Activated Killer), MHC non-restricted T cells or NKT cells are used. A method of measuring the radioactivity of ⁵¹ Cr that has been destroyed by the action and released, and further labeled with a non-radioactive europium (Eu) -DTPA chelate that does not use a radioactive substance, and this is labeled with NK, LAK cells, MHC non-restricted A fluoroimmunoassay for the so-called europium release test and an enzyme immunoassay for the LDH method, in which Eu-DTPA destroyed and released by T cells or NKT cells is measured by a time-resolved fluorescence method, are known.
[0003]
However, in measuring these immunity, the lymphocyte culture medium is simply prepared from the collected blood and its immunity is evaluated, and attention is paid only to the improvement of the apparatus for efficiently carrying out these measurement methods. It was only.
[0004]
[Problems to be solved by the invention]
The present invention is a method for quantitatively measuring the effects of various activators on lymphocyte immunity and the effects of cancer cytotoxic substances added to a solution of target cancer cells by applying a conventional method for measuring immunity. In addition to establishing a system for computer processing and utilizing the vast amount of data obtained, it is intended to help improve the immunity of healthy people as well as treating cancer patients.
[0005]
[Means for Solving the Problems]
In the immunopotentiation measurement method of the present invention, first, when measuring the immunopotency by a cytotoxic activity test, pretreatment of adding an immunopotentiator or cytotoxic substance to a lymphocyte solution separated from blood And a lymphocyte culture medium.
[0006]
Here, the immunopotentiator was selected from various cytokines, various biological response modifiers (BRM), useful substances contained in various herbal medicines or various foods, or trace metal elements that affect intracellular machinery. These are one type or a mixture of two or more types, and their immunity can be evaluated generally or individually.
[0007]
Second, in order to measure immunity directly useful for diseases such as cancer treatment and viral pathogens, for example, when immunity is measured by a cytotoxic activity test, for example, damage of cancer cells against target cell solutions. A pretreatment for adding a cancer cytotoxic substance that facilitates the treatment to obtain a target cancer cell treatment solution, and the treatment solution is added to a lymphocyte medium separated from blood to measure the immunity of the lymphocytes. A method for measuring immune capacity was proposed. The cancer cytotoxic substance in this case is an anticancer drug, an anticancer drug, a cancer suppressor contained in food or a substance that activates immune ability. Cancer cytotoxic substances refer to all substances that selectively damage cancer cells compared to general cells. For example, in addition to substances called anticancer drugs and anticancer drugs, anticancer effects have been discovered. All drugs are subject to this. The cancer therapeutic effect of these substances can be quantitatively determined.
[0008]
Although there are various cytotoxic activity tests, the present invention can preferably be used for a release test with a radioisotope ⁵¹ Cr in which the cytotoxic activity test is part of a fluoroimmunoassay or a europium release test or radioimmunoassay. Thus, the immunoassay method for determining% cytotoxic activity of a stock solution or a plurality of dilution series samples in which the number of lymphocytes and the number of target cells (TC) in various lymphocyte media are in a predetermined ratio.
[0009]
Various cytokines used here include IL-1 to 18, TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ, GCSF, M-CSF, and GM-CSF.
[0010]
Examples of various biological response modifiers (BRM) include OK432 (Pisibanil, Chugai Pharmaceutical registered trademark), Shizofilan (Sonifilan, Kaken Pharmaceutical registered trademark), Urinaftatin (Miraclide, Mochida Pharmaceutical registered trademark), Lentinan, Maruyama vaccine, and the like.
[0011]
In addition, various herbal medicines include the following herbal medicines: Wei Sensen, 茵 ▲ Chen ▼ 蒿, 茴香, 延胡索, 黄耆, 黄 ▲ 今 ▼, 黄柏, 黄連, 遠志, 薤 白, 艾葉, 首 首烏, Kakkon, talc, turf root, kanrohito, pomegranate, licorice, bellflower, chrysanthemum, coconut, cypress, apricot kernel, ginseng, persimmon, katsura, safflower, red rice, katsuki, kobok, ox knee, wu Salmon, Beef Eggplant, Sesame, Gomiko, Saiko, Spicy, Yam, Yam, Soy Sauce, Herbal Medicine, Shiso, Ginseng, Carpiko, Shredded Sandwich, Ginger, Wheat, Sesame, Sea bream, hot pepper, divine song, plaster, river ▲ bow ▼, sea bream, mulberry white skin, Daihuang, Daegu, Sawan, ground yellow, bamboo grove, earth bone skin, acquaintance mother, Chotou, sea bream, Chen skin, tempura, winter lion , Toki, Taojin, Independence, Ginseng, Mumon Winter, Huanghe, Semi-Summer, White ▲ Stop ▼, White Birch, Eggplant, Sakai, Shujitsuko, Self-prevention, Windproof, I ▲ Bundle ▼, Peony Skin, Oyster, Mao , Asakojin, incense, kidori, etc. These are also available as single or mixed products, and are available as Mao-to, Katsue-yu, Daijoki-yu, Shirato-yu, Dai-kotsuji-yu, Yotsuka-to, Koshiba-ko-yu, Shishiko Hot water, Hochuekki hot water, Rikkunshi hot water, Juzendai hot water, Ginseng Yoei hot water, etc. can also be evaluated as activators.
[0012]
In addition, various foods include cereals and legume rice, barley rice wheat, barley, pearl barley, buckwheat, corn, soybeans, red beans, peas, kidney beans, strawberries and starches, sugar and sweeteners, seasonings, There are many kinds of beverages such as spices, fats and milks, seeds, seafood, poultry and eggs, vegetables, fruits, mushrooms, seaweeds. In addition to these, AHCC, agaricus, chitin chitosan, royal jelly, propolis, shark cartilage, liver extract of anko as well known as vitamins and health foods. Also included in the foods are folk medicines such as Amachazuru, Aloe, Ikarisou, Kashiwa, and Dokudami.
[0013]
Trace elements that affect intracellular machinery include elements that form so-called inorganic salts, ash, and minerals that have important physiological functions on cells, and are Ca, P, K, S, Na. , Cl, Mg, Fe, Cu, Mn, I, Al, Zn, Se, Ge, and the like.
[0014]
Various herbal medicines, various foods and the like are generally dried by a method that does not damage the ingredients, or powdered and extracted with water or other solvent, and used as an extract that is concentrated if necessary.
[0015]
Substances that facilitate cancer cell damage are known as various anti-cancer agents, and the effects of any substance that has been described so far on the immunity of lymphocytes can be examined by the method of the present invention. .
[0016]
The relationship between the% cytotoxic activity obtained by these methods for measuring immunity and the immunopotentiator is computer-processed by the following means. That is, the immune capacity evaluation system of the present invention processes by the following means.
1. Data measured by the fluorometer is transmitted to a computer via a communication line and stored in a temporary storage device of the computer.
2. A specific number is assigned to the data and stored in the storage device.
3. The data stored in the mass storage device is called into the temporary storage device, edited into a print format, and the% cytotoxic activity is calculated.
[0017]
According to the method of the present invention, it is possible to evaluate the performance of various activators that activate lymphocytes to enhance immunity and to evaluate the effects of cytotoxic substances such as anticancer agents. Moreover, there is an advantage that judgment of a patient's case, state, etc. can be quantified by automatically collecting analysis data.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail by way of examples. However, these are only a part of an enormous amount of materials and are not limited to the examples.
[0019]
First, as a first step, a lymphocyte separation protocol as an effector will be described. Blood collected from humans is placed in a centrifuge tube, diluted twice with phosphate buffer solution (PBS), transferred to a plurality of lymphoprep tubes for blood separation, centrifuged at room temperature, and the lymphocyte layer is centrifuged. Collect in a tube, wash with PBS, centrifuge and discard the supernatant. After repeating this operation three times, the plate was further washed twice with PBS at a low temperature, the RPMI of the culture solution was added, the concentration was measured, and the concentration was adjusted to 4 × 10 ⁶ lymphocytes / ml. This concentration measurement was performed using a hemocytometer and was measured as accurately as possible to obtain a lymphocyte culture stock solution. This stock solution was diluted 5 times, 10 times, 20 times, and 40 times as necessary. These adjusted liquids are called lymphocyte culture adjustment liquids. Up to this point, the lymphocyte culture medium has been conventionally adjusted by a similar method, and the method has been arranged.
[0020]
A feature of the present invention resides in that a lymphocyte culture solution obtained as described above is further treated by adding various immunopotentiating substances to a novel lymphocyte culture medium. That is, 0.1 ml of the above-mentioned lymphocyte culture preparation solution was dispensed into a plurality of tubes, an equal amount of RPMI was added as a blank, and many activators shown in Table 1 were changed in concentration to other tubes. An amount was added, placed on a tube stand, stored in a CO ₂ incubator, and cultured overnight to prepare a sample for measuring immunity by a cytotoxicity test.
[0021]
[Table 1]

[0022]
Next, as a second step, a general immunopotentiation measurement cell K562 labeling protocol as a target cell (target cell, TC) will be described. For cell preparation, weigh 5 mg of dextran sulfate into a 1.5 ml centrifuge tube, add K562 to 5 × 10 ⁶ , centrifuge at 4 ° C., remove the supernatant, and replace buffer A. After repeating twice, the labeling buffer by Eu-DTPA was adjusted. This is done in standard steps. The labeling substance can be replaced with a label using radioactive chromium 51 instead of europium. In this method, chromium may be added to the target cells and cultured in a CO ₂ incubator at 37 ° C. for 1 hour. In addition, instead of K562, cancer cells of the individual to be examined can be taken out and labeled, and in this case, it is possible to accurately select an immunity stimulating substance to be administered to each individual.
[0023]
0.5 ml of the prepared labeling buffer was placed in a plurality of tubes, cooled on ice, added with buffer B, stirred, and then centrifuged and the supernatant removed twice. By this operation, the inside of the cell was stabilized and used as a target cell (TC).
[0024]
The target cells were further added with 2 ml of RPMI, pipetted and stirred, and then centrifuged at 4 ° C. and removal of the supernatant three times in the same manner as described above, and the cancer cells K562 that had been refrigerated for 1-2 hours. The number of cells was counted and diluted with RPMI so as to be 5 × 10 ⁴ / ml to obtain a target cell solution. Then, mitomycin C, an anticancer agent shown in the lower part of Table 1, was added to this solution as a cancer cytotoxic substance and pretreated to obtain a target cancer cell treatment solution.
[0025]
The cytotoxic activity test solution was prepared by mixing the target cell solution with the lymphocyte culture stock solution described in the first step described above at a predetermined ratio, and used for measurement of lymphocyte immunity. Therefore, the number of cells was adjusted so that the ratio of cancer cells: lymphocytes was always the same. For example, a dilution series sample of 5: 1 to 10: 1, 20: 1, 40: 1, etc. with 200 cells per 1,000 lymphocytes was tested.
[0026]
The immunity can be measured by the above-mentioned known cytotoxic activity test. Here, the release test with the radioisotope ⁵¹ Cr (Cr method) or the further release test with the europium element (Eu method) is performed. Most appropriate. The specific testing method was based on a method established as a radioimmunoassay for the Cr method and a fluoroimmunoassay for the Eu method.
[0027]
First, cancer cells are labeled with the radioisotope ⁵¹ Cr. The target cells (TC) are mixed with lymphocytes that are effectors at an appropriate ratio, and ⁵¹ Cr that appears in the supernatant when the target cells are damaged is measured with a gamma counter. Since this method uses radioactive materials, there is a need for special facilities, there are problems of radioactive waste disposal and exposure to ⁵¹ Cr that emits radiation, and Europium (Eu) -DTPA has excellent correlation with this method. It is easier to carry out by the chelate method. This is called the europium release test. The europium release test uses target cells in which cancer cells are labeled with Eu-DTPA chelate, destroys the target cells with MHC non-restricted T cells or NKT cells by NK (Natural Killer) and LAK (Lymphokine Activatid Killer) cells. The amount of released Eu-DTPA is measured by a time-resolved fluorescence method. Since the specific method of the release test is known, the details are omitted, but the europium release test is particularly suitable for the present invention.
[0028]
As described above, the cancer cell is the target cell (TC) with the radioactive orientation element ⁵¹ Cr, and lymphocytes as effectors are mixed at an appropriate ratio, so that when the target cell is injured, it appears in the supernatant. Measure ⁵¹ Cr with a gamma counter, or mix target cells labeled with Eu-DTPA with effector lymphocytes and measure Eu in the supernatant with a fluorometer. A block diagram of the system is shown in FIG. Each europium (Eu) release test sample is first recorded and recorded as a specimen name, and after preparing plate data, the measurement data is collected and recorded by measurement with a fluorometer.
[0029]
That is, the computer processing of the measurement data is performed in the following order. In the fluorometer, data measured by the trade name Delphia 1234 fluorometer or Arbo 1420 fluorometer is transmitted to the computer via the CCITT V24 interface, and the measurement data is stored in a temporary storage device of the computer. A specimen name is registered in advance for the data, a specific number is assigned, and the data is stored in the mass storage device together with the data. The measurement data accumulated in the large-capacity storage device is called into the temporary storage device and used as measurement graph data, which is edited into a print format to calculate% cytotoxic activity.
[0030]
Specimen name registration here refers to registration of the specific name of the activator to the disk device. For example, in the case of the cytokine interleukin IL2 or Chinese herbal medicine, registration of substance names such as Shosaikoto and Hochuekkito do. Plate data clearly indicates which of the above-mentioned specimens is in which position of the fluorometer measurement container, and this is also registered in the disk device as described above, and the plate data list is confirmed.
[0031]
Next, the measurement is performed with a fluorometer. Here, a Delphia 1234 fluorometer from WALLAC is used to measure the number of Eu and collect the data. In other words, data indicating the percentage of the target cells labeled with Eu-DTPA and the percentage S / M ratio between “spontaneous Eu3 + release” (S) and “maximum Eu3 + release” (M) is generated as% cytotoxic activity. And graph it.
[0032]
When target cells by Eu-DTPA are used, a flowchart of the measurement with “Delphia Eu-labeling kit” of Pharmacia Biotechnology is shown in FIG. That is, the measurement data editing screen is roughly divided into a “measurement data collection screen”, a “measurement data list display screen”, a “plate data creation screen”, a “plate data list display screen”, and a “specimen name registration screen”. Further, these screens shift to the work items (1) to (5), respectively.
[0033]
FIG. 2 is a basic creation flowchart of measurement data, and shows basic functions for performing measurement work. Here, the work items are simply displayed as (1) to (5), but the work item (1) on the measurement data recording screen is the mainstream, and (2) to (5) are auxiliary functions of (1). Therefore, it will be omitted and further explanation will be given for (1).
[0034]
FIG. 3 is a continuation flow from (1) in FIG. 2, and is a data collection flowchart from the fluorometer Delphia. Enter the subject (patient) number, name, and plate data while viewing the measurement data storage screen, create a measurement data file, create a spreadsheet file, run the spreadsheet program, and move to the next flow.
[0035]
FIG. 4 is a flowchart for calculating the% cytotoxic activity from the measurement data, and shows the internal processing for the spreadsheet that is continued from (6) in FIG. FIG. 5 is a measurement print format creation flowchart, and shows the continuation work items from (9) in FIG. Here, the printing format is determined, and a flow of printing and printing out a graph and a data table is shown.
[0036]
Here, the measurement results obtained with respect to the effect of the various activators of FIG. 1 described above on the% cytotoxic activity will be specifically described. Table 2 shows the immunity at the time of blood collection in% cytotoxicity with respect to 1,000 lymphocytes described above diluted to a ratio of 5: 1, 10: 1, 20: 1 and 40: 1 of 200 cells. The value was 10.4-34.1.
[0037]
[Table 2]

[0038]
Table 3 shows interleukin 2 (IL2) as a cytokine in Table 1 added to lymphocytes, and OK432 (Bisibanil) as BRM was added to lymphocytes to examine their% cytotoxic activity. Obtained high numbers of 53.8-96.3% and 42.4-84.4%.
[0039]
[Table 3]

[0040]
The attack power against cancer cells of lymphocytes in the collected blood is expressed as injury activity value, and the ratio is the relationship between the number of lymphocytes and cancer cells. The case where 40 lymphocytes attack is shown. Therefore, comparing the results in Tables 2 and 3, it can be shown that the interleukin 2 can be enhanced to about 2.8 to 5.2 times with respect to the immunity at the time of blood collection, and about 2.5 to 4.1 times with OK432. Understand.
[0041]
Table 4 shows the results obtained by calculating the% cytotoxic activity by the method of the present invention for 5 types such as Sho-saiko-to from among the main formulas of Sanyin Sanyo, a Chinese herbal medicine, in the same manner as cytokines. From this result, it can be said that if the subject takes Hochuekkito, the immunity can be increased to about 2.2 to 2.9 times the immunity at the time of blood collection, and the immunity in the other 4 types. It can be seen that it is useful for evaluating the aggressiveness of lymphocytes against cancer cells for these activators.
[0042]
[Table 4]

[0043]
Table 5 shows the measurement results when chitosan and agarics as useful substances contained in food as activators, aluminum as a trace element, and anticancer agent (mitomycin C) as a cytotoxic substance are used.
[0044]
[Table 5]

[0045]
From the results in Table 5, in this subject, when chitosan was taken, the immunity increased by about 10% compared to the immunity at the time of blood collection, but in Agaricus, it increased by less than about 5%. Chitosan and Agaricus show that it is more efficient to take chitosan. In addition, although the negative effects of aluminum have been screamed for a long time now, it is expected that the immune capacity will be reduced to about 80% compared to the immune capacity at the time of blood collection, so if this subject needs to increase the immune capacity It can be said that it is better not to use aluminum. Next, when mitomycin C, an anticancer agent, is administered to a living body, in addition to its ability to damage its own cancer cells, the anticancer agent efficiently converts cancer cells into the patient's immune system. The ability to injure is also recognized. That is, in this case, it is expected that the patient's lymphocytes can injure the cancer cells about twice as much as when nothing is administered, and it can be seen that the administration of this anticancer agent is effective for the patient. Moreover, although an anticancer effect is reported also about vitamin C, if a patient takes vitamin C, immunity will be expected to increase about 1.5 times, which indicates that there is a benefit for the patient.
[0046]
FIG. 6 to FIG. 8 are graphs based on the numerical data shown in Tables 2 to 5 so that the effect of each activator can be easily understood. FIG. 6 is a bar graph showing the% cytotoxic activity of interleukin 2, picibanil and 5 kinds of Chinese herbal medicines. Interleukin 2, picibanil and Hochuekkito have the effect on the activation of blood immunity of this subject. It turns out that it is very effective. It is also possible to determine what has little or no effect. FIG. 7 shows that the subject's immunity is considerably high, so a large increase cannot be expected as in the subject of FIG. 6, but a slight increase is seen in chitosan and agaricus. Although there is no significant penalty for aluminum, it can be seen that there is a considerable decrease in aluminum and it is better not to use it. FIG. 8 shows that the subject's blood immunity is as low as 21.7 and is a patient with advanced cancer, but mitomycin C administration and vitamin C administration are useful means for eliminating cancer in the living body.
[0047]
【The invention's effect】
As described above, according to the immunological capacity measurement method and the evaluation system thereof of the present invention, conventionally, immunological capacity measurement methods such as radioimmunoassay and fluoroimmunoassay have been used only for measuring the immunological capacity of human blood. Although it has not been applied to the determination of quantitative values for the effects of immunopotentiators and, conversely, cytotoxic substances, it has become possible to quantify the efficacy of these substances. As a result, it was possible to give accurate guidance on dietary care and drug administration to each subject, and these treatment effects could be judged by quantification of numerical values and continuous measurement.
[0048]
In the present invention, the completion of a system that can manage the measurement method by computer makes it possible to quickly and reliably evaluate blood lymphocyte activators and anticancer substances.
[0049]
In particular, it seems to be effective in treating cancer patients, but it can also be used for general immunity measurement tests aimed at improving the immunity of not only cancer patients but also healthy people. In other words, the measures to activate the lymphocytes possessed by the subject themselves to increase immunity depend on the stimulating substance, but this method is originally based on food or an active ingredient in food, so drugs such as anticancer agents Compared to, there are few side effects. It is an extremely safe treatment. The present invention is an extremely useful method for measuring immunity and its evaluation system in that the application of this point can be advanced in a wide range in institutions such as hospitals and health centers.
[Brief description of the drawings]
FIG. 1 is a block diagram of an immune capacity automatic measurement system.
FIG. 2 is a flowchart for creating basic measurement data.
FIG. 3 is a data collection flowchart from measurement Delphia.
FIG. 4 is a flowchart for calculating% cytotoxic activity from measured data.
FIG. 5 is a measurement print format creation flowchart.
FIG. 6 is a graph showing changes in immunity by each stimulating substance.
FIG. 7 is a graph showing changes in immunity by each stimulating substance.
FIG. 8 is a graph showing changes in immunity caused by cytotoxic substances and vitamin C.

Claims (5)

Radioimmunoassay, enzyme immunoassay, or by cytotoxic activity test fluoro immunoassay upon measuring the immunity, to a solution of lymphocytes isolated from blood, various cytokines, various biological response modifiers as activator of immune competence (BRM ), and the lymphocyte medium was treated before the addition of one or a mixture of two or more selected from among trace metals affecting useful substances or intracellular machinery contained in various herbal or various foods The target cell solution is mixed with the lymphocyte medium at a predetermined ratio to obtain a sample for measuring immunity, and the target is a sample having a predetermined ratio between the number of lymphocytes and the number of target cells (TC) in various lymphocyte culture media. immunity measuring method comprising Rukoto seek cytotoxic activity. An anticancer drug or anticancer drug that is a cytotoxic substance of a cancer cell as a cytotoxic substance against a lymphocyte solution separated from blood in the measurement of immunity by a cytotoxic activity test of radioimmunoassay, enzyme immunoassay or fluoroimmunoassay Pretreatment by adding a drug to obtain a lymphocyte culture medium, and a target cell solution is mixed with the lymphocyte culture medium at a predetermined ratio to prepare a sample for measuring immunity. The number of lymphocytes in each lymphocyte culture medium and target cells (TC) immunity measuring method and number and said Rukoto seek% cytotoxic activity targeting the sample to be a predetermined ratio. Various cytokines and various biological response modifiers as stimulators of immune capacity that facilitate cell damage against target cell solutions when measuring immunogenicity by radioimmunoassay, enzyme immunoassay or fluoroimmunoassay cytotoxicity test (BRM), treatment of target cells by pretreatment by adding one or two or more mixtures selected from useful substances contained in various herbal medicines or various foods or trace metal elements that affect intracellular machinery And the treatment solution is added to a lymphocyte medium separated from blood to measure the immunity of the lymphocytes, and the number of lymphocytes and the number of target cells (TC) in each lymphocyte medium are in a predetermined ratio. A method for measuring immunity , characterized in that% cytotoxic activity is determined for a sample . Radioimmunoassay, when measuring the immunity by cytotoxicity test of an enzyme immunoassay or fluoro immunoassay, to a solution of the target cell, the addition of an anti-cancer agent or anticancer agent Ru injury substance der cancer cells as cytotoxic agents To prepare a target cell treatment solution, and add the treatment solution to a lymphocyte medium separated from blood to measure the immunity of the lymphocytes. The number of lymphocytes in each lymphocyte medium and target cells (TC) ) A method for measuring immunity , characterized in that% cytotoxic activity is determined for a sample whose number is a predetermined ratio . A series of operations are automatically computer-processed by the following means with respect to the relationship between the% cytotoxic activity obtained by the method for measuring immunity according to any one of claims 1 to 4 and an immunopotentiator. Immune capacity evaluation system.
1. Data measured by the fluorometer is transmitted to a computer via a communication line and stored in a temporary storage device of the computer.
2. A specific number is assigned to the data, and the data is stored in the storage device.
3. The data stored in the storage device is called into the temporary storage device, edited into a print format, and the% cytotoxic activity is calculated.

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