JPH01224400A - Monoclonal antibody recognizing human arteriosclerosis and its production - Google Patents
Monoclonal antibody recognizing human arteriosclerosis and its productionInfo
- Publication number
- JPH01224400A JPH01224400A JP63050162A JP5016288A JPH01224400A JP H01224400 A JPH01224400 A JP H01224400A JP 63050162 A JP63050162 A JP 63050162A JP 5016288 A JP5016288 A JP 5016288A JP H01224400 A JPH01224400 A JP H01224400A
- Authority
- JP
- Japan
- Prior art keywords
- monoclonal antibody
- arteriosclerosis
- human
- human arteriosclerosis
- recognizing
- 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
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Abstract
Description
【発明の詳細な説明】
産業上のｆ用
本発明は、ヒト動脈硬化症関連抗原を特異的に認識する
ヒト動脈硬化症認識モノクロナル抗体およびその製造法
に関する。さらに本発明は、ヒト動脈硬化症関連抗原の
定量およびそれに基づく動脈硬化症の診断、ヒト動脈硬
化症の病巣に対するイメージング診断などに有用なヒト
動脈硬化症関連抗原認識モノクロナル抗体を提供するも
のである。DETAILED DESCRIPTION OF THE INVENTION For industrial use The present invention relates to a human arteriosclerosis-recognizing monoclonal antibody that specifically recognizes a human arteriosclerosis-related antigen and a method for producing the same. Furthermore, the present invention provides a monoclonal antibody that recognizes human arteriosclerosis-related antigens, which is useful for quantifying human arteriosclerosis-related antigens, diagnosing arteriosclerosis based on the determination, and imaging diagnosis of human arteriosclerosis lesions. be.
ｉ の ・および；題
動脈硬化症（Ａ ｒＬｅｒｉｏｓｃｌｅｒｏｓｉｓ）は
大動脈、冠動脈、脳動脈および脈動脈等の筋型動脈に多
く発生し、狭心症、心筋梗塞、脳梗塞等の主因となる疾
患である。Arteriosclerosis (ArLeriosclerosis) is a disease that often occurs in muscular arteries such as the aorta, coronary arteries, cerebral arteries, and pulsatile arteries, and is the main cause of angina pectoris, myocardial infarction, and cerebral infarction. .
その病因として血漿コレステロールの上昇、内皮傷害、
血小板ａ２集、内膜肥厚、粥腫の形成等が提唱されてい
る。しかしその成因はほとんど解析されていないのが現
状である。Its pathogenesis includes elevated plasma cholesterol, endothelial damage,
Platelet A2 collection, intimal thickening, atherogenesis, etc. have been proposed. However, at present, its causes have hardly been analyzed.
正常な大動脈は内皮、弾性繊維と平滑筋細胞よりなる中
膜、弾性繊維よりなる外股の３層により構築されている
。この大動脈が何らかの原因でこの内皮と中膜の境界が
肥厚し、細胞の異常繁殖、壊死が生ずるといわゆる動脈
硬化症として出現する。A normal aorta is composed of three layers: the endothelium, the tunica media made of elastic fibers and smooth muscle cells, and the tunica media made of elastic fibers. When the boundary between the endothelium and the tunica media of the aorta thickens for some reason, abnormal cell proliferation and necrosis occur, so-called arteriosclerosis occurs.
すなわち、心筋梗塞、脳梗塞等の重篤な疾患が生ずる原
因として、
■ コレステロールをはじめとする脂質の動脈壁細胞な
らびに細胞間への蓄積による粥腫形成、■ 細胞増殖に
伴う内膜肥厚および
■ 内皮の損傷ならびに内股の肥厚に伴う血小板の凝集
等の種々の理由により動脈血管の閉塞がもたらされるこ
とである。その結果、狭心症、心筋梗塞、脳梗塞等の疾
患を発現するものと推定される。In other words, the causes of serious diseases such as myocardial infarction and cerebral infarction include: ■ atherogenesis due to the accumulation of cholesterol and other lipids in arterial wall cells and between cells, ■ intimal thickening due to cell proliferation, and ■ Arterial blood vessel occlusion results from various reasons such as endothelial damage and platelet aggregation due to medial thickening. As a result, it is estimated that diseases such as angina pectoris, myocardial infarction, and cerebral infarction occur.
上記内膜肥厚部分には
イ） 大量の脂質を取り込んだ泡沫細胞の出現、口）
細胞間への脂質の蓄積、
ハ） 内膜で゛の平滑筋細胞の増殖、
二）結き組織の増成とカルシウムの沈着、ホ）血小板の
凝集と血栓形成、
等が認められる。In the thickened area of the intima, a) appearance of foam cells that have taken in large amounts of lipids;
Accumulation of lipids between cells, c) proliferation of smooth muscle cells in the intima, b) growth of connective tissue and calcium deposition, e) aggregation of platelets and thrombus formation, etc. are observed.
従来、ヒト動脈硬化症の診断法としては、血中コレステ
ロール値の測定、リボ蛋白組成の分析、凝固因子の検索
などから危険度を予測する間接的な方法と、動脈壁の音
波の伝搬速度や反射エコーを利用して動脈硬化の進展度
を測定したり、動脈血管内に造影剤を注入して動脈の侠
窄像を直接観察する方法とがある。Traditionally, methods for diagnosing human arteriosclerosis include indirect methods that predict the risk based on measurements of blood cholesterol levels, analysis of riboprotein composition, and searches for coagulation factors; There are methods to measure the progress of arteriosclerosis using reflected echoes, and methods to directly observe the image of arterial stenosis by injecting a contrast medium into the arterial blood vessel.
しかしながら、上記の間接法における血中危険因子の測
定は動脈硬化症の直接因子を測定しているものではなく
、確度の高い診断法とは言えない。However, the above-mentioned indirect method for measuring blood risk factors does not measure direct factors for arteriosclerosis, and cannot be said to be a highly accurate diagnostic method.
一方、反射エコー法、血管造影法による直接診断法はい
ずれも動脈硬化症による血管の狭窄度を測定する方法で
あり、動脈硬化の進展度そのものを測定する方法ではな
い。しかも血管造影法は造影剤を動脈内に注入するため
に技術的な危険性を伴う。On the other hand, direct diagnostic methods using reflection echo method and angiography are both methods for measuring the degree of stenosis of blood vessels due to arteriosclerosis, and are not methods for measuring the degree of progression of arteriosclerosis itself. Moreover, angiography involves technical risks because a contrast medium is injected into the artery.
従って、手軽にでき、かつヒト動脈硬化症に特異性の高
い診断法が望まれている。しかしながら動脈硬化症を詮
所するための直接的な血中指標物質ならびに動脈硬化巣
を直接認識する指標物質は何ら見出されていないのが現
状である。Therefore, a diagnostic method that is easy to perform and highly specific for human arteriosclerosis is desired. However, at present, no indicator substance in the blood that directly detects arteriosclerosis or an indicator substance that directly recognizes arteriosclerotic lesions has been found.
５題を 史 るための
本発明は前述した問題点を解決するものであり、本発明
者等は、ヒト動脈硬化症に直接作用する因子について鋭
意研究した結果、動脈硬化症患者からの血清または動脈
硬化病巣部位を抗原として、家族性高脂血症、心筋梗塞
、脳梗塞等のヒト動脈硬化症の関連抗原を特異的に認識
するモノクロナル抗体産生細胞を単離し、この細胞から
ヒト動脈硬化症認識モノクロナル抗体を得ることに成功
した。The present invention is intended to solve the above-mentioned problems, and as a result of intensive research into factors that directly affect human arteriosclerosis, the present inventors have found that blood serum or We isolated monoclonal antibody-producing cells that specifically recognize antigens related to human arteriosclerosis such as familial hyperlipidemia, myocardial infarction, and cerebral infarction using arteriosclerotic lesions as antigens, and used these cells to detect human arteriosclerosis. We succeeded in obtaining a monoclonal antibody that recognizes the disease.
すなわち本発明は、ヒト動脈硬化症関連抗原を特異的に
認識するヒト動脈硬化症認識モノクロナル抗体に関する
ものである。さらに本発明は、ヒト動脈硬化症関連抗原
を含有する溶液で、ヒトを除く哺乳動物を免疫し、該動
物め抗体産生リンパ球とミエローマ細胞とを細胞融合さ
せて抗ヒト動脈硬化症抗体産生融合細胞を単離し、次い
で抗ヒト動脈硬化症抗体産生融合細胞を培養することを
特徴とするヒト動脈硬化症認識モノクロナル抗体の製造
法に関する。That is, the present invention relates to a human arteriosclerosis-recognizing monoclonal antibody that specifically recognizes a human arteriosclerosis-related antigen. Furthermore, the present invention involves immunizing a mammal other than a human with a solution containing a human arteriosclerosis-related antigen, and fusing the animal's antibody-producing lymphocytes with myeloma cells to produce an anti-human arteriosclerosis antibody. The present invention relates to a method for producing a monoclonal antibody that recognizes human arteriosclerosis, which comprises isolating cells and then culturing fused cells that produce anti-human arteriosclerosis antibodies.
本発明のモノクロナル抗体は、好適な簡便法として、抗
ヒト動脈硬化症抗体産生細胞とミエローマ細胞との融合
細胞から産生することができる。The monoclonal antibody of the present invention can be produced from a fused cell of an anti-human arteriosclerosis antibody-producing cell and a myeloma cell, as a suitable and simple method.
この製造法は特に限定されるものではなく、例えば、ま
ずヒト動脈硬化症抗原を用いて常法によりヒトを除く哺
乳動物を感作する。ここで用いうるヒト動脈硬化症抗原
としては、例えば家族性高脂血症患者血清や脳梗塞、心
筋梗塞患者血清のようなヒト動脈硬化症患者から分離し
た血清および動脈硬化病巣部位（特に内股肥厚部分）の
ホモゲネートを挙げることができる。血清で感作する場
き、健康人のヒト正常血漿で感作して得た抗体をヒト動
脈硬化症患者の血清と混合し、この混合物を用いて動物
を感作することが好しい。この混合物の使用によって正
常血漿由来の抗原に対する抗体産生を抑え、患者血清由
来の抗原に対する産生の確率を高めることができる、と
いう利点がある。ついで感作された動物の肺臓、胸線、
末梢リンパ節、または末梢血より抗ヒト動脈硬化症抗体
産生のリンパ球を単離して抗ヒト動脈硬化症抗体産生細
胞を得る。さらにこの細胞は、ミエローマ細胞と常法に
より融合させ、抗体産生融合細胞を得る。この融合細胞
を複数のウェルに分注し、培養し、各ウェルの上清を酵
素免疫測定法（ＥＬＩＳＡ法〉、あるいは間接オートラ
ジオグラフィー法等の手段により分析しヒト動脈硬化患
者血清あるいはヒト動脈硬化病巣にのみ特異的に反応し
、正常ヒト血清あるいは正常動脈壁とは反応しないヒト
動脈硬化症認識モノクロナル抗体産生細胞を単離する。This production method is not particularly limited; for example, first, mammals other than humans are sensitized using a human arteriosclerosis antigen by a conventional method. Human arteriosclerosis antigens that can be used here include serum isolated from human arteriosclerosis patients, such as serum from patients with familial hyperlipidemia, serum from patients with cerebral infarction or myocardial infarction, and arteriosclerotic lesions (particularly those from inner thigh thickening). (part) homogenate. When sensitizing with serum, it is preferable to mix antibodies obtained by sensitizing with normal human plasma from healthy individuals with serum from human arteriosclerosis patients, and use this mixture to sensitize the animal. The use of this mixture has the advantage of suppressing the production of antibodies against antigens derived from normal plasma and increasing the probability of production against antigens derived from patient serum. The lungs and thorax of the sensitized animal were then
Anti-human arteriosclerosis antibody-producing lymphocytes are isolated from peripheral lymph nodes or peripheral blood to obtain anti-human arteriosclerosis antibody-producing cells. Furthermore, this cell is fused with myeloma cells by a conventional method to obtain an antibody-producing fused cell. The fused cells were dispensed into multiple wells, cultured, and the supernatant of each well was analyzed by means such as enzyme-linked immunosorbent assay (ELISA) or indirect autoradiography. Cells producing human arteriosclerosis-recognizing monoclonal antibodies that specifically react only with sclerotic lesions and do not react with normal human serum or normal arterial walls are isolated.
モノクロナル抗体を用いて動脈硬化病巣部位画像解析を
行なう場合、モノクロナル抗体により動脈硬化病巣の表
層をより特異的に認識することが望ましい。前記の間接
オートラジオグラフィー法で分析することによって、動
脈硬化病巣の表層部を特異的に認識する抗体産生細胞を
単離することができるというところに特徴を有する。さ
らにこのモノクロナル抗体産生細胞を直接組織培養す°
るか、または哺乳動物、例えばマウスやモルモットの腹
腔に移植して腫瘍を形成させて腹水から産生された目的
とするモノクロナル抗体を採取し、精製する。When performing image analysis of arteriosclerotic lesions using a monoclonal antibody, it is desirable to more specifically recognize the surface layer of the arteriosclerotic lesion using the monoclonal antibody. The method is characterized in that antibody-producing cells that specifically recognize the surface layer of arteriosclerotic lesions can be isolated by analysis using the indirect autoradiography method described above. Furthermore, these monoclonal antibody-producing cells can be directly cultured in tissues.
Alternatively, the antibody is transplanted into the peritoneal cavity of a mammal, such as a mouse or a guinea pig, to form a tumor, and the monoclonal antibody of interest produced from ascites is collected and purified.
かくして、本発明者等は、ヒト動脈硬化症関連抗原を特
異的に認識する新規ヒト動脈硬化症認識モノクロナル抗
体を単離することに成功した。Thus, the present inventors succeeded in isolating a novel human arteriosclerosis-recognizing monoclonal antibody that specifically recognizes a human arteriosclerosis-related antigen.
このうち、高脂血症患者、脳梗塞患者、心筋梗塞患者血
清を抗原として得られたモノクロナル抗体１３１Ｂ等は
ヒト思考血清に認められる動脈硬化症関連抗原を特異的
に認識するモノクロナル抗体で、免疫グロブリンクラス
はＩｇＧに分類される。Among these, monoclonal antibody 131B, which was obtained using serum from patients with hyperlipidemia, cerebral infarction, and myocardial infarction, is a monoclonal antibody that specifically recognizes arteriosclerosis-related antigens found in human serum. , the immunoglobulin class is classified as IgG.
更に、動脈硬化症患者の内膜肥厚部分のホモゲネートを
抗原としてモノクロナル抗体１２５Ｈを得ることができ
た。このモノクロナル抗体１２５Ｈは免疫グロブリンク
ラスＩｇＧに分類されるもので、動脈硬化巣を特異的に
認識していることが示された。Furthermore, monoclonal antibody 125H could be obtained using a homogenate of the intimal thickened area of an arteriosclerosis patient as an antigen. This monoclonal antibody 125H is classified as an immunoglobulin class IgG, and was shown to specifically recognize arteriosclerotic lesions.
これらのモノクロナル抗体１３１Ｂおよび１２５Ｈはい
ずれもヒト患者血清あるいは動脈壁ホモゲネートを抗原
として６作し作製したもので、家兎血清ならびに動脈壁
ホモゲネートを抗原として作製したモノクロナル抗体と
は異なる新規なものであった。These monoclonal antibodies 131B and 125H were both produced using human patient serum or arterial wall homogenate as antigens, and are new and different from monoclonal antibodies produced using domestic rabbit serum and arterial wall homogenate as antigens. Met.
さらにこのモノクロナル抗体は、必要に応じてそのまま
、またはその蛋白分解酵素処理による分解産物であるＦ
（ａｂ’ ＞２．Ｆａｂとしてヒト動脈硬化症の進展等
を測定する試薬として用いることができる。ちなみに、
このモノクロナル抗体の認識する抗原は、ヒト動脈硬化
症の病巣部位のみならず患者血清中にも存在している。Furthermore, this monoclonal antibody can be used as it is or as a degradation product obtained by treatment with a protease.
(ab'> 2. As a Fab, it can be used as a reagent to measure the progression of human arteriosclerosis. Incidentally,
The antigen recognized by this monoclonal antibody is present not only at the focal site of human arteriosclerosis but also in patient serum.
それゆえ採取した血清を、モノクロナル抗体を反応試薬
として、例えばアルカリホスファターゼ、β−ガラクト
シダーゼ、パーオキシダーゼ等の酵素、放射性同位元素
、蛍光物質等の認識物質を用いた種々の免疫測定法、例
えば競合法またはサンドイツチ法や′ａ集反応または凝
集阻止反応による測定法またはそれらの改良測定法等公
知の方法により、その抗原を定量または定性し、その結
果から動脈硬化の進展度を判断することができるもので
ある。Therefore, the collected serum is subjected to various immunoassay methods, such as competition, using monoclonal antibodies as reaction reagents, enzymes such as alkaline phosphatase, β-galactosidase, and peroxidase, and recognition substances such as radioisotopes and fluorescent substances. The antigen can be quantified or qualitatively determined by a known method such as the Sand-Deutsch method, the aggregation reaction or aggregation inhibition reaction, or an improved measurement method thereof, and the degree of progression of arteriosclerosis can be determined from the results. It is something.
さらにまたこのモノクロナル抗体を用いて、ヒト動脈硬
化症の病巣部位の存在または広がりを検査することがで
きる。即ち放射性同位元素、例えばＮａ’コ’ Ｉ 、
ＮａＩ２コ■やＮａ１２’Ｉ等を用いクロラミンＴ法、
酵素法等にてヨードを、又５ｎＣ１２等還元剤の存在下
にＮａ””ＴｃＯ，（過テクネチウム酸ナトリウム）生
理食塩液を加えることによりテクネチウムを、又適当な
キレート剤（無水ＤＴＰＡ ＋Ｄ １ｅｔｈｙｌｅｎｅ
Ｔｒｉａｍｉｎｅ Ｐｅｎｔａ Ａｃｅｔｉｃ ａｃ
ｉｄ等の所謂Ｂ １ｒｕｎｃｔｉｏｎａｌキレート）を
介しインジウムＣ１１■ｎ）を上記のモノクロナル抗体
又はそのＦ（ａｂ’）ｚ、Ｆａｂフラグメント等に結合
させる。Furthermore, this monoclonal antibody can be used to examine the presence or spread of focal areas of human arteriosclerosis. i.e. radioactive isotopes, such as Na'co'I,
Chloramine T method using NaI2 or Na12'I,
Iodine is added by an enzymatic method, technetium is added by adding Na""TcO, (sodium pertechnetate) physiological saline in the presence of a reducing agent such as 5nC12, and technetium is added by an appropriate chelating agent (anhydrous DTPA + D1ethylene).
Triamine Penta Acetic ac
Indium C11■n) is bonded to the above monoclonal antibody or its F(ab')z, Fab fragment, etc. via a so-called B1runctional chelate such as id, etc.
これを無菌無毒性媒体に加え静脈内に投与し、−定時間
後に病巣部位に結合した結果をガンマカメラ等を用いて
体内の放射能分布に基づいたシンチグラムを得る。この
シンチグラムより、脳梗塞、心筋梗塞等のヒト動脈硬化
症の病巣部位の存在または広がりを検査することができ
るものである。This is added to a sterile, non-toxic medium and administered intravenously, and after a certain period of time it binds to the lesion site, and a scintigram based on the distribution of radioactivity in the body is obtained using a gamma camera or the like. Using this scintigram, it is possible to examine the presence or spread of focal areas of human arteriosclerosis such as cerebral infarction and myocardial infarction.
実」１凹−
次いで本発明の実施例を挙げるが、本発明は何らこれに
よって限定されるものではない。Examples of the present invention will be described below, but the present invention is not limited thereto in any way.
（１） モノクロナル抗体の作製
正常者血漿の１名分を２０μ！取り、１８０μｌの生理
食塩水にて１０倍希釈し、Ｂ Ａ Ｌ Ｂ ／ ｃマウ
スに腹腔的投与した。さらに３週間後と２ケ月後に同様
の投与を行い、抗正常者血漿抗血清を得た。同時に、家
族性高脂血症患者血清の１９名分を混和しこの２０μｌ
を、８０μｌの生理食塩水にて５倍希釈した。これに１
００μｌのフロイント完全アジュバントを加えてよく混
和し、得られたエマルジョンをＢＡ Ｌ Ｂ ／ ｃマ
ウスに皮下投与した。２週間後に上記抗正常者血漿抗血
清１０μｌと、家族性高脂血症患者１９名の混合血清１
０μｌとを８０μｌの生理食塩水で希釈後、フロイント
完全アジュバント１００μｌを加えてよく混和し、得ら
れたエマルジョンを先のマウスに皮下投与した。さらに
最終免疫として２週間後に、抗正常者血漿抗血清１０μ
ｌと家族性高脂血症患者１９名の混合血清１０μｌを１
８０μｌの生理食塩水で希釈し腹腔内投与後、３日日に
肺臓を取り出した。(1) Preparation of monoclonal antibodies 20μ of plasma from a normal person! The solution was diluted 10 times with 180 μl of physiological saline and intraperitoneally administered to BALB/c mice. After another 3 weeks and 2 months, the same administration was performed to obtain anti-normal plasma antiserum. At the same time, mix 19 familial hyperlipidemia patient serum and add 20μl of this.
was diluted 5 times with 80 μl of physiological saline. 1 for this
00 μl of Freund's complete adjuvant was added and mixed well, and the resulting emulsion was subcutaneously administered to BALB/c mice. Two weeks later, 10 μl of the above anti-normal plasma antiserum and mixed serum 1 of 19 familial hyperlipidemia patients were added.
After diluting 0 μl with 80 μl of physiological saline, 100 μl of complete Freund's adjuvant was added and mixed well, and the resulting emulsion was subcutaneously administered to the mouse. Furthermore, as a final immunization, 2 weeks later, 10μ of anti-normal plasma antiserum was administered.
10 μl of mixed serum from 19 patients with familial hyperlipidemia.
After dilution with 80 μl of physiological saline and intraperitoneal administration, the lungs were removed on the 3rd day.
胛細胞はＨＨＢＳ（ＨＥＰＥＳ Ｂｕｆｆｅｒｅｄｌ
（ｎｎｋｓ’Ｂａ１ａｎｃｅｄ ５ａｌｔ ５ｏｌｎ、
）にてよく洗浄し、同様に洗浄したマウスミエローマ細
胞株Ｐ３／Ｕ１と５：１の割きで混合し、１３００ｒｐ
ｍで５分間遠心した。沈査として得られた細胞を５０％
ポリエチレングリコール４０００を含むＤ’ＭＥＭ（−
）培地ＩＭｌに浮遊させ２分間放置した。次いで、Ｄ’
ＭＥＭ（−）培地１０ｚＮ！を徐々に加えて希釈後、８
００ｒｐ情にて５分間遠心した。次いで得られた細胞を
２０％の牛胎児抗血清を含む）（ＡＴ培地１００Ｍ１に
懸濁し、０．１ｍｅずつを９６ウエルの組織培養プレー
トに分注した。The caterpillar cells are HHBS (HEPES Bufferedl)
(nnks'Ba1anced 5alt 5oln,
), mixed with mouse myeloma cell line P3/U1 washed in the same manner at a ratio of 5:1, and incubated at 1300 rp.
Centrifuged for 5 minutes at m. 50% of the cells obtained as sediment
D'MEM containing polyethylene glycol 4000 (-
) The cells were suspended in medium IMl and left for 2 minutes. Then D'
MEM(-) medium 10zN! After diluting by gradually adding 8
Centrifugation was performed for 5 minutes at 00 rpm. Then, the obtained cells were suspended in 100 M1 AT medium (containing 20% fetal bovine antiserum), and 0.1 me portions were dispensed into a 96-well tissue culture plate.
２〜４日毎に半量ずつ培地を交換し、８日後の培養上清
について抗体価を調べたところ、１０００ウエル中２１
ウエルで強い抗体活性が見られた０次いで限界希釈法に
よりクローニングを行い、正常者１４名の混合血漿及び
１４名の混合血清では抗体活性を示さず、ヒト血清中の
動脈硬化症関連抗原を特異的に認識する株を選別したと
ころ計９株の融合細胞が得られた。さらに最終的に限界
希釈法によりクローニングを行い、より正常者血漿及び
血清で゛は活性を示さず、動脈硬化症関連疾患の患者血
清に特異的に反応する融合細胞を計６株単離した。When the culture medium was replaced by half every 2 to 4 days and the antibody titer was examined for the culture supernatant after 8 days, 21 out of 1000 wells were detected.
Strong antibody activity was observed in the well. Cloning was then performed by limiting dilution method, and mixed plasma of 14 normal subjects and mixed serum of 14 subjects did not show antibody activity, indicating that the antibody was specific for arteriosclerosis-related antigens in human serum. As a result, a total of nine fused cell lines were obtained. Finally, cloning was carried out by the limiting dilution method, and a total of six fused cells were isolated that showed no activity in plasma and serum from normal subjects but specifically reacted with serum from patients with arteriosclerosis-related diseases.
これらをブリスタンで処理されたＢ Ａ Ｌ Ｂ ／
ｃマウスの腹腔内に注入し、１０〜２０日後にその腹水
を採取してモノクロナル抗体を得た。BAL B /
c It was injected intraperitoneally into a mouse, and the ascites was collected 10 to 20 days later to obtain a monoclonal antibody.
この６株の融合細胞のうち、１３１Ｂ細胞株から得られ
たモノクロナル抗体は、オフタロニー法による免疫グロ
ブリンクラスは図１に示すようにＩｇＣ；＋に分類され
るもので、ヒト血清中動脈硬化症関連抗原を特異的に認
識するモノクロナル抗体である。１３１Ｂ細胞株は微生
物工業技術研究所特許微生物寄託センター（以下、微工
研と略称する）にＦＥＲＭ ＢＰ−１６７６として寄
託されている。Among these six fused cell lines, the monoclonal antibody obtained from the 131B cell line is classified as IgC;+ in the immunoglobulin class by the Ophthalony method, as shown in Figure 1, and is associated with human serum arteriosclerosis. It is a monoclonal antibody that specifically recognizes a related antigen. The 131B cell line has been deposited as FERM BP-1676 at the Patent Microorganism Depositary Center of the National Institute of Microbial Technology (hereinafter abbreviated as FERM).
（２）１３１Ｂモノクロナル抗体による患者血清の測定
被検血清として、家族性高脂血症患者血清（ＦＨ）、■
型高脂血症患者血清（Ｔ ｙｐｅ ｍ ＞、低βリポタ
ンパク血症患者血清（Ｈｙｐｏβ）、心筋梗塞患者血清
（ＭＩ＞、脳梗塞患者血清（ＡＰＯ）、ウニルナ−症候
群患者血清（Ｗｅｒｎｅｒ＞、狭心症患者血清（Ａ Ｂ
ｉｎａ）、正常者血漿（ＮＰ）、正常者１４名より調製
した混合血清（ＮＳ）の１０００倍希釈液１００μｌを
、ＥＬＩＳＡ用マイクロプレート（Ｆ ａｌｃｏｎ ３
９１２）に加え、４℃にて一晩放置し吸着させた。１％
ＢＳＡを含む燐酸ｗｉ液でマイクロプレートを処理後、
１３１Ｂモノクロナル抗体溶液を１００μｌ添加し、３
７℃にて２時間反応させた０次いで洗浄後、１０，００
０倍希釈したアルカリフォスファターゼ標識抗マウスＩ
ｇＧ （ｔａｇｏ社製）を１００μｔ’添加して３７
℃で１時間反応させ、洗浄後、１１ｇ／ｍｌバラニトロ
フェニルリン酸２ナトリウム塩、０．０１％ＭｇＣ１２
を含む１ｍＭジェタノールアミンＭ街液（ｐＨ９，８）
を１００１□１加え、３７℃にて２時間反応させた。マ
イクロプレート用比色計（バイオ・ラット社製）を用い
吸光度（ＯＤ４．５ｎｍ）を測定し動脈硬化症関連抗原
活性値とした。その結果を、第１表に示す０本発明の１
３１Ｂモノクロナル抗体は、家族性高脂血症患者血清（
ＦＨ）、■型高脂血症患者血清（Ｔｙｐｅｌ［Ｉ）、低
βリポタンパク血症患者血清（Ｈｙｐｏβ）、心筋梗塞
患者血清（Ｍ Ｉ ＞、脳梗塞患者血清（ＡＰＯ）、ウ
ニルナ−症候群患者血清（Ｖ ｅ ｒ ｎ ｅ ｔ ）
、狭心症患者血清（Ａｎｇｉｎａ）の動脈硬化症関連抗
原を強く認識し高い値を示した。しかも正常者血漿（Ｎ
Ｐ）及び血清（ＮＳ）に対する値は小さいことが明らか
であり、このモノクロナル抗体はヒト血清中の動脈硬化
症関連抗原を特異的に認識したものであると判断できる
。(2) Measurement of patient serum using 131B monoclonal antibody As test serum, familial hyperlipidemia patient serum (FH), ■
Type hyperlipidemia patient serum (Type m>, hypoβ lipoproteinemia patient serum (Hypoβ), myocardial infarction patient serum (MI>, cerebral infarction patient serum (APO), Unirna syndrome patient serum (Werner>, Angina patient serum (A B
100 μl of a 1000-fold dilution of normal plasma (NP), mixed serum (NS) prepared from 14 normal subjects was placed in an ELISA microplate (Falcon 3
912) and allowed to stand overnight at 4°C to allow adsorption. 1%
After treating the microplate with phosphoric acid solution containing BSA,
Add 100 μl of 131B monoclonal antibody solution and
After 2 hours of reaction at 7°C and washing, 10,000
Alkaline phosphatase-labeled anti-mouse I diluted 1:0
37 by adding 100 μt' of gG (manufactured by Tago).
After reacting at ℃ for 1 hour and washing, 11 g/ml balanitrophenyl phosphate disodium salt, 0.01% MgC12
1mM jetanolamine M street solution (pH 9,8) containing
1001□1 of was added and reacted at 37°C for 2 hours. Absorbance (OD 4.5 nm) was measured using a microplate colorimeter (manufactured by Bio-Rat) and used as an arteriosclerosis-related antigen activity value. The results are shown in Table 1.
31B monoclonal antibody was obtained from familial hyperlipidemia patient serum (
FH), type II hyperlipidemia patient serum (Type [I), hypoβlipoproteinemia patient serum (Hypoβ), myocardial infarction patient serum (M I >, cerebral infarction patient serum (APO), Uniruna syndrome patient serum Serum (Vernet)
, strongly recognized arteriosclerosis-related antigens in angina patient serum (Angina) and showed high values. Moreover, normal plasma (N
It is clear that the values for P) and serum (NS) are small, and it can be concluded that this monoclonal antibody specifically recognizes an arteriosclerosis-related antigen in human serum.
以上の結果を第２図にまとめた。第２図から明らかなよ
うに、家族性高脂血症患者、■工高脂血症患者、低βリ
ポタンパク血症患者、心筋梗塞患者および脳梗塞患者血
清はいずれも正常者血漿及び血清よりも高い値を示した
。The above results are summarized in Figure 2. As is clear from Figure 2, serum from familial hyperlipidemia patients, industrial hyperlipidemia patients, hypoβlipoproteinemia patients, myocardial infarction patients, and cerebral infarction patients were all obtained from normal plasma and serum. also showed high values.
（３）１３１Ｂモノクロナル抗体と反応するヒト血清動
脈硬化症関連抗原物質
家族性高脂血症患者血清をＳＤＳ添加ポリアクリルアマ
イドゲルにて４０Ｖ、−晩電気泳動しニトロセルロース
膜にブロッティングした。この膜を、０．０５％非イオ
ン系界面活性剤ツイーン２０を含む燐酸＆！街液にてよ
く洗浄後、５％ＢＳＡを含む燐酸緩衝液にて一晩処理し
た。洗浄後、１３１Ｂモノクロナル抗体を２時間、アル
カリフォスファターゼ標識抗マウスＩｇＧ抗体を１時間
反応させた０反応後、０．２％の５−ブロモ−４−クロ
ロ−３−インドリルリン酸パラトルイジン塩を含む０．
７５Ｍトリス−塩酸緩衝液（ｐＨ８，８）の溶液に膜を
浸し、室温にて一晩放置し免疫染色した。その結果、１
３１Ｂモノクロナル抗体の認識するヒト血清動脈硬化症
関連抗原物質の分子量は約５２ 、０００と推定される
。(3) Human serum arteriosclerosis-related antigen substance reacting with 131B monoclonal antibody Familial hyperlipidemia patient serum was subjected to electrophoresis at 40 V overnight on an SDS-added polyacrylamide gel and blotted onto a nitrocellulose membrane. This film was coated with phosphoric acid containing 0.05% nonionic surfactant Tween 20! After thoroughly washing with street solution, the plate was treated with a phosphate buffer containing 5% BSA overnight. After washing, 131B monoclonal antibody was reacted for 2 hours and alkaline phosphatase labeled anti-mouse IgG antibody was reacted for 1 hour. After 0 reaction, 0.2% 5-bromo-4-chloro-3-indolyl phosphate paratoluidine salt was added. Including 0.
The membrane was immersed in a solution of 75M Tris-HCl buffer (pH 8.8) and left overnight at room temperature for immunostaining. As a result, 1
The molecular weight of the human serum arteriosclerosis-related antigen recognized by the 31B monoclonal antibody is estimated to be approximately 52,000.
また、１３１Ｂ抗体が認識する抗原物質は、動脈硬化症
と密接な関係にあると言われているリボ蛋白の構成成分
ＡｐｏＡ Ｉ 、Ａ−ｆｆ、Ｂ −１００，Ｂ −４
８゜Ｅ、Ｄなどとは異なる高脂血症特異抗原物質である
（第３図参照）。In addition, the antigenic substances recognized by the 131B antibody are riboprotein constituents ApoA I, A-ff, B-100, and B-4, which are said to be closely related to arteriosclerosis.
It is a hyperlipidemia-specific antigen substance different from 8°E and D (see Figure 3).
さらに本抗体の認識する抗原物質は図４に示すように、
家族性高脂血症患者血清ＩＲ１をＮ ａ Ｂ ｒにて比
重を１．２５に合わせ、その上に比重１．１５３の生理
食塩水を２ｚｌ、さらに比重１．０６３の生理食塩水１
．６ｄ重層し、３００００ｒｐｍ２４時間の条件下で遠
心しても浮上しない画分に存在する。しかも図５に示す
ように家族性高脂血症患者のコレステロール値との相関
が小さい（Ｒ＝０．１０４）ことから、この１３１Ｂモ
ノクロナル抗体はＬＤＬをはじめとする、リボ蛋白質を
抗原物質として認識しないことが確認された。Furthermore, the antigenic substances recognized by this antibody are as shown in Figure 4.
The serum IR1 of a patient with familial hyperlipidemia was adjusted to a specific gravity of 1.25 with NaBr, and then 2 zl of physiological saline with a specific gravity of 1.153 was added, and then 1 ml of physiological saline with a specific gravity of 1.063 was added.
．． It exists in a fraction that does not float even after centrifugation at 30,000 rpm for 24 hours. Furthermore, as shown in Figure 5, the correlation with cholesterol levels in patients with familial hyperlipidemia is small (R = 0.104), so this 131B monoclonal antibody uses riboproteins such as LDL as antigens. It was confirmed that it was not recognized.
以上の結果より、１３１Ｂモノクロナル抗体は動脈硬化
症血清中に特徴的に共通して存在する抗原物質を認識し
ていると結論される。From the above results, it is concluded that the 131B monoclonal antibody recognizes an antigen substance characteristically and commonly present in arteriosclerotic serum.
また１３１Ｂモノクロナル抗体は血漿成分が混入しても
、実質上反応に影響を与えないことが判っている。Furthermore, it has been found that even if the 131B monoclonal antibody is contaminated with plasma components, the reaction is not substantially affected.
第１表
１３１ Ｂモノクロナル抗体による動脈硬化関連疾患Ｔ
、Ｃ，：総コレステロール
ＦＨ：家族性高脂血症
ＡＰＯ：＆ｉ梗塞
ＭＩ ：心筋梗塞
Ｈｙｐｏβ：低βリボ蛋白血症
Ｔｙｐｅｍ：ｍ型高脂血症
ｗｅｒｎｅｒ ：ウエルｆ Ｍ 候ＩＴＡＢｉｎａ
：狭心症
２ 動 硬 病巣を として；製した（１）抗原の調
製方法
ヒト胸部及び腹部大動脈硬化壁を摘出し、冷所にて血管
を切開したのちビンセットにて内膜肥厚部（動脈硬化病
巣）を剥離し、ハサミにて１１角に細切後、１ ｍＭ
Ｅ Ｄ Ｔ Ａ 、０．１％エタノールを含む水溶液（
ｐＨ７，４＞を湿重量１ｇ当り５〜１０ｍ１の割合で加
え、ポリトロンホモゲナイザー（ｐｏｌｙｔｒｏｎｈｏ
ｍｏＦｌｅｎｉｚｅｒ）にてホモゲネー） （ｈｏｍｏ
ｇｅｎａｔｅ）を調製した。Table 1 131 Arteriosclerosis-related disease T caused by monoclonal antibody B
, C,: Total cholesterol FH: Familial hyperlipidemia APO: &i Infarction MI: Myocardial infarction Hypoβ: Hypoβ-riboproteinemia Type: M-type hyperlipidemia werner: Wel f M symptom ITABina
: Angina pectoris 2 arteriosclerotic lesions; (1) Preparation method of antigen Extract the sclerotic wall of human thoracic and abdominal aorta, incise the blood vessel in a cold place, and remove the intimal thickened area (arterial thickening) using a bottle set. After peeling off the sclerotic lesions and cutting them into 11-sided pieces with scissors, 1 mM
EDTA, aqueous solution containing 0.1% ethanol (
pH 7.4> was added at a ratio of 5 to 10 ml per 1 g of wet weight, and
homogenized with moFlenizer) (homo
Generate) was prepared.
このホモゲネート３名（５検体）分の混合物を４重のガ
ーゼでヂ過し、ｒ液を抗原液とした。This homogenate mixture for 3 people (5 samples) was passed through 4 layers of gauze, and the r solution was used as an antigen solution.
（２）モノクロナル抗体の作製−
ホモゲネート０．５ｚｇ（２００μｉ）に２００μｌの
フロイント完全アジュバントを加えてよく混和した。得
られたエマルジョンをＢＡＬＢ／ｃマウスの皮下に投与
した。９週後に同様の投与を行い、さらに１８週後に最
終免疫として０．５Ｂ（２００μｌ）のホモゲネートに
Ｄ’ＭＥＭ（−）培地２００Ａｔ１を加えたちのを腹腔
内投与し、３日目に肺臓を取り出した。これをＤ’ＭＥ
Ｍ（−）にてよく洗浄し、同様に洗浄したマウスミエロ
ーマ細胞株Ｐ３／Ｕｌと５：１の割合で混合し、ＩＱＯ
Ｏｒｐ−で５分間遠心した。(2) Preparation of monoclonal antibody - 200 μl of Freund's complete adjuvant was added to 0.5 zg (200 μi) of the homogenate and mixed well. The obtained emulsion was administered subcutaneously to BALB/c mice. After 9 weeks, the same administration was carried out, and after 18 weeks, as a final immunization, 0.5B (200 μl) of homogenate plus 200 At1 of D'MEM (-) medium was administered intraperitoneally, and on the 3rd day, the lungs were removed. Ta. D'ME this
Wash thoroughly with M(-), mix with similarly washed mouse myeloma cell line P3/Ul at a ratio of 5:1, and add IQO.
Centrifugation was performed in Orp- for 5 minutes.
沈さとして得られた細胞を４５％のポリエチレングリコ
ール４０００を含むＤ’ＭＥＭ（−）培地１ｘｌに浮遊
させ２分間放置した０次いで、Ｄ’ＭＥＭ（−）培地１
０ｚｉを徐々に静かに加えて希釈後、１０００ｒｐ＋＊
にて５分間遠心した０次いで得られた細胞を１０％の牛
胎児血清を含むＤ’ＭＥＭ培地１２７ｚｊ！に懸濁し、
１１１ずつを２４ウエルの組織培養プレートに分注した
。The cells obtained by sedimentation were suspended in 1xl of D'MEM (-) medium containing 45% polyethylene glycol 4000 and left for 2 minutes.
After diluting by gradually and gently adding 0zi, 1000rp+*
The cells were centrifuged for 5 minutes at 100 ml of D'MEM medium containing 10% fetal bovine serum. suspended in
111 portions were dispensed into 24-well tissue culture plates.
１日目にＨＡＴ培地１１１を加え、１〜２日毎に半量ず
つ培地交換を行い、８日後の培養上清の抗体価をＥＬＩ
ＳＡ法で調べた。Add HAT medium 111 on the first day, replace the medium by half every 1 to 2 days, and measure the antibody titer of the culture supernatant after 8 days by ELI.
I investigated using the SA method.
抗原として前記のヒト病巣内膜肥厚部のホモゲネートを
１０μｇ／ｍｌに調製し、１００μｌをＥＬＩＳＡ用マ
イクロプレート（タンク社製）に加え、４℃にて一晩放
置して吸着させた。２％ＢＳＡ溶液でマイクロプレート
を処理後、１２７ウエルの培養上清を１００μ！添加し
て室温にて２時間反応させた。As an antigen, the homogenate of the human lesion intimal thickening was prepared at 10 μg/ml, 100 μl was added to an ELISA microplate (manufactured by Tank Co., Ltd.), and the antigen was allowed to stand overnight at 4° C. for adsorption. After treating the microplate with 2% BSA solution, add 100μ! of culture supernatant from 127 wells. The mixture was added and reacted at room temperature for 2 hours.
次いで洗浄後、ａ、ｏｏｏ倍希釈したアルカリフォスフ
ァターゼ標識抗マウス１．Ｇ抗体（フジ社製）を１００
μ！添加して室温で２時間反応させ、洗浄後、１ａｇ／
ｘｉバラニトロフェニルリン酸２ナトリウム塩、０．０
１％ＭｇＣ１，を含む１ｍＭジェタノールアミンＭ衝液
（ｐＨ９，８＞を１００μＩ加え、室温にて１時間反応
させた。マイクロプレート用比色計（バイオ・チック社
Ｗ）を用い吸光度（ＯＤ 、。ｇｎｍ）を測定した。吸
光度０．５以上の６５ウエルについてさらにオートラジ
オグラフィー法で検討した。After washing, a, alkaline phosphatase-labeled anti-mouse 1. G antibody (manufactured by Fuji) 100
μ! After adding and reacting at room temperature for 2 hours, after washing, 1ag/
xi Balanitrophenyl phosphate disodium salt, 0.0
100 μl of 1mM jetanolamine M solution (pH 9.8>) containing 1% MgCl was added and reacted for 1 hour at room temperature. Absorbance (OD) was measured using a microplate colorimeter (Bio-Chick Co., Ltd. W). gnm) was measured. 65 wells with absorbance of 0.5 or higher were further examined by autoradiography.
１％コレステロール添加食で４ケ月飼育した動脈硬化症
モデルウサギの動脈硬化病巣を取り出し、６５の小片に
切断する。これを５％ＢＳＡ溶液で処理後、６５ウエル
の培養上清を１１１１添加して４℃で５時間反応させた
０次いで洗浄後、５％正常ヤギ血清で処理し、２００μ
ｌ（１，７μＣｉ／ｚｊりの１２５工標識抗マウスＩｇ
Ｇ抗体く非標識体：フジ社製）を添加して４℃、３時間
反応させた。洗浄後、Ｘ−ｒａｙフィルム（フジ社製）
を用いオートラジオグラフィーを行った。Arteriosclerosis lesions from arteriosclerosis model rabbits fed a diet supplemented with 1% cholesterol for 4 months were removed and cut into 65 small pieces. After treating this with 5% BSA solution, 1111 culture supernatant of 65 wells was added and reacted at 4°C for 5 hours. After washing, it was treated with 5% normal goat serum and 200 μl of culture supernatant was added.
l (1.7 μCi/zj 125-labeled anti-mouse Ig
An unlabeled G antibody (manufactured by Fuji Co., Ltd.) was added and reacted at 4°C for 3 hours. After washing, apply X-ray film (manufactured by Fuji)
Autoradiography was performed using
この中から特異的な反応を示し、結合率の高い１０ウエ
ルを選択し、限界希釈法によりクローニングを行った。From among these, 10 wells that showed a specific reaction and a high binding rate were selected and cloned using the limiting dilution method.
その結果、計２株の融合細胞が単離された。これらをプ
リスタンで処理されたＢＡＬＢ ／ ｅマウスの腹腔内
に注入し、１０〜１６日後にその腹水を採取してモノク
ロナル抗体を得た。この２株の融合細胞のうち、１２５
Ｈ細胞株から得られた１２５Ｈモノクロナル抗体は、Ｅ
ＬＩ ＳＡ法による免疫グロブリンクラスはＩｇＧ、ｂ
に分類されるもので、動脈硬化壁内膜肥厚部を特異的に
認識するモノクロナル抗体である。この１２５Ｈ細胞株
は微工研にＦＥＲＭ ＢＰ−１６７５として寄託された
。As a result, a total of two fused cell lines were isolated. These were injected intraperitoneally into BALB/e mice treated with pristane, and their ascites fluid was collected 10 to 16 days later to obtain monoclonal antibodies. Of these two fused cells, 125
The 125H monoclonal antibody obtained from the E.
Immunoglobulin class according to LISA method is IgG,b
It is a monoclonal antibody that specifically recognizes the intimal thickening of the arteriosclerotic wall. This 125H cell line was deposited with the Microtech Institute as FERM BP-1675.
（３）１２５Ｈモノクロナル抗体の特異性蛋白量１０μ
ｇ／ａｌの調製した色々なヒト病巣内膜肥厚部および正
常動脈内膜のホモゲネート１００μｌを用い、上記ＥＬ
ＩＳＡ法にて１２５Ｈモノクロナル抗体の反応性を検討
した。その結果を第２表及び第６図に示すが、このモノ
クロナル抗体はヒト動脈硬化症病巣と強い反応性を示す
ことが確認できた。(3) Specificity protein amount of 125H monoclonal antibody 10μ
The above-mentioned EL
The reactivity of the 125H monoclonal antibody was examined using the ISA method. The results are shown in Table 2 and Figure 6, and it was confirmed that this monoclonal antibody showed strong reactivity with human arteriosclerosis lesions.
なお、総コレステロール（ＴＣ）値の測定は、コレステ
ロール測定キット（Ｖ−コレスターゼ「ニツスイ」：口
承製薬社製）を用いた。In addition, the total cholesterol (TC) value was measured using a cholesterol measurement kit (V-cholestase "Nitsui", manufactured by Oral Pharmaceutical Co., Ltd.).
また、この１２５Ｈモノクロナル抗体のウサギの動脈硬
化内膜肥厚部との反応性を検討した。１％コレステロー
ル食で飼育した動脈硬化症モデルウサギの動脈硬化病巣
の凍結切片を用いた間接蛍光抗体法による免疫染色およ
びオイルレッド０による染色の様子をそれぞれ第７図お
よび第８図に示す。Furthermore, the reactivity of this 125H monoclonal antibody with arteriosclerotic intimal thickening areas in rabbits was investigated. Immunostaining by indirect fluorescent antibody method and staining with Oil Red 0 using frozen sections of arteriosclerosis lesions of arteriosclerosis model rabbits fed a 1% cholesterol diet are shown in FIGS. 7 and 8, respectively.
また１％コレステロール食で飼育した動脈硬化症モデル
ウサギの動脈硬化病巣を取り出し、上記オートラジオグ
ラフィー法にて１２５Ｈモノクロナル抗体の反応性を検
討した０間接オートラジオグラフィー法による反応の様
子および病巣の様子をそれぞれ第９図および第１０図に
示す。In addition, arteriosclerotic lesions were removed from arteriosclerosis model rabbits fed a 1% cholesterol diet, and the reactivity of 125H monoclonal antibodies was examined using the above-mentioned autoradiography method. The situation is shown in FIG. 9 and FIG. 10, respectively.
図から明らかなように１２５Ｈモノクロナル抗体はウサ
ギ動脈硬化内膜肥厚部を特異的に認識した。As is clear from the figure, the 125H monoclonal antibody specifically recognized the intimal thickened area of arteriosclerosis in rabbits.
従って、本１２５Ｈモノクロナル抗体は体内投与に際し
ても病巣と反応することが期待される。また、ヒト動脈
硬化症診断のための画像解析用試薬を調製し、ヒトに応
用するに際し、ウサギを用いて投与量、安全試験等の予
備試験を十分に行うことができるものであり、ヒト動脈
硬化巣の部位特定や広がりを調べるための画像解析等へ
の応用が期待される。Therefore, the present 125H monoclonal antibody is expected to react with lesions even when administered in the body. In addition, when preparing image analysis reagents for diagnosing human arteriosclerosis and applying them to humans, it is possible to sufficiently conduct preliminary tests such as dosage and safety tests using rabbits. It is expected to be applied to image analysis to identify the location and spread of sclerotic lesions.
（４） １２５８モノクロナル抗体と反応する動脈硬
化壁抗原物質
病巣肥厚内膜および正常動脈壁のホモゲネートをＳＤＳ
添加ポリアクリルアマイドゲルにて２０１ＩＡ、２時間
電気泳動し、さらにニトロセルロース膜に電気的にブロ
ッティングした。この膜を、０．０５％非イオン系界面
活性剤ツイーン２０を含む燐酸Ｍ衝液にてよく洗浄後、
２％ＢＳＡ溶液にて６時間処理した。洗浄後、１２５Ｈ
モノクロナル抗体と１６時間、アルカリフォスファター
ゼａ識抗マウスＩｇＧ＋ＩｇＭ抗体と２時間反応させた
０反応後０．１％の５−ブロモ−４−クロロ−３−イン
ドリルリン酸パラトルイジン塩を含む０．７５Ｍ ）リ
スー塩酸Ｍｆｆｌ液（ｐＨ８，８）の溶液に膜を浸し、
室温にて２時間放置した。その結果、１２５Ｈモノクロ
ナル抗体が認識する抗原物質の分子量は約４４．Ｇｏｏ
であることが確認された（第１１図参照）。(4) Atherosclerotic wall antigen that reacts with the 1258 monoclonal antibody. Homogenates of the thickened intima and normal arterial walls were collected using SDS.
Electrophoresis was performed on a polyacrylamide gel containing 201IA for 2 hours, followed by electroblotting onto a nitrocellulose membrane. After thoroughly washing this membrane with a phosphoric acid M solution containing 0.05% nonionic surfactant Tween 20,
It was treated with a 2% BSA solution for 6 hours. After washing, 125H
After reacting with a monoclonal antibody for 16 hours and with an alkaline phosphatase A-recognizing mouse IgG+IgM antibody for 2 hours, a solution containing 0.1% 5-bromo-4-chloro-3-indolyl phosphate paratoluidine salt was added. Immerse the membrane in a solution of 75M) lithium-hydrochloric acid Mffl solution (pH 8,8),
It was left at room temperature for 2 hours. As a result, the molecular weight of the antigenic substance recognized by the 125H monoclonal antibody was approximately 44. Goo
It was confirmed that (see Figure 11).
第２表
病変名・・・ ＦＰ：ファイブラスプラークＦＳ：ファ
ッティストリークス
ＡＰ：アテロマタスプラーク
ＣＯ：石灰化
Ｎ ：正常
判定基準・・・ＥＬＩＳＡ（ＯＤ、。５）の値による０
、８以上 ：４＋
０．６〜Ｇ、８：３＋
０．４〜０．６：２＋
０．２〜０．４：＋
０．２以下 二 −
Ｃｈｏｌｅｓｔｅｒｏｌ／ ｐ、（コレステロール／タ
ンパク質）＝ （ｍｇ／ ｄ／）／ （贋ＩＦ／ Ｉ／
）Table 2 Lesion name: FP: Fibrous plaque FS: Fatty streaks AP: Atheromatous plaque CO: Calcification N: Normality criteria: 0 based on ELISA (OD, .5) value
, 8 or more: 4+ 0.6-G, 8: 3+ 0.4-0.6: 2+ 0.2-0.4: + 0.2 or less 2 - Cholesterol/p, (cholesterol/protein) = (mg / d/)/ (fake IF/ I/
)
第１ｒＭはオフタロニー法による１３１ Ｂモノクロナ
ル抗体の免疫グロブリンクラスを示す写真である。
第２図は第１表に示した１３１Ｂモノクロナル抗体の疾
患側抗原活性をプロットした図である。
第３図は１３１Ｂモノクロナル抗体が認識する動脈硬化
関連物質をタンパク染色及び免疫染色によって示す図で
ある。
第４図は１３１Ｂモノクロナル抗体の認識する抗原物質
比重を示す図である。
第５図は１３１Ｂモノクロナル抗体の認識する抗原物質
と家族性高脂血症患者のコレステロール値との相関を示
す図である。
第６図は第２表に示した１２５Ｈモノクロナル抗体の疾
患別抗原活性をプロットした図である。
第７図及び第８図は１２５Ｈモノクロナル抗体と反応さ
せたウサギの動脈硬化病巣の凍結切片を用いたそれぞれ
間接蛍光抗体法による免疫染色及びオイルレッドによる
染色の様子を示す写真である。
第９図及び第１０図はウサギの動脈硬化病巣に対する１
２５Ｈモノクロナル抗体の間接オートラジオグラフィー
法による反応の様子及び病巣の様子をそれぞれ示す写真
である。
第１１図は１２５Ｈモノクロナル抗体が認識する抗原物
質をタンパク染色及び免疫染色によって示す写真である
。
１３１Ｂモノクロ一ナル伍体疾患別伍原活性）６ ろ
１ン］
笛４図
ｄ＝１．０６３ − ’７ラタンヨン吟号 ｄ
＝１．２５０第 ケ記
垂龜・コレフチＯ−Ｉし （ｍｇ／ｄｆ ）１４図
１２５Ｈモノクロナル抗体
′）１１ ノ ≧【ｊ
・￥Ｊ 罰
づ 、−Ｉ ＼：、１１The first rM is a photograph showing the immunoglobulin class of 131 B monoclonal antibody by Ophthalony method. FIG. 2 is a diagram plotting the disease-side antigen activity of the 131B monoclonal antibody shown in Table 1. FIG. 3 is a diagram showing arteriosclerosis-related substances recognized by the 131B monoclonal antibody by protein staining and immunostaining. FIG. 4 is a diagram showing the specific gravity of antigenic substances recognized by the 131B monoclonal antibody. FIG. 5 is a diagram showing the correlation between the antigenic substance recognized by the 131B monoclonal antibody and the cholesterol level of familial hyperlipidemia patients. FIG. 6 is a diagram plotting the disease-specific antigenic activity of the 125H monoclonal antibody shown in Table 2. FIGS. 7 and 8 are photographs showing immunostaining by indirect fluorescent antibody method and staining with oil red, respectively, using frozen sections of rabbit arteriosclerotic lesions reacted with 125H monoclonal antibody. Figures 9 and 10 show 1
These are photographs showing the state of reaction and the state of lesions obtained by indirect autoradiography of 25H monoclonal antibody. FIG. 11 is a photograph showing the antigenic substance recognized by the 125H monoclonal antibody by protein staining and immunostaining. 131B monochrome monochromatic body disease-specific Gohara activity) 6 Ro 1] Flute 4 figure d = 1.063 - '7 Ratan Yong Gingo d
= 1.250 No. KEKI SUPPLY/KOLEFT O-I (mg/df) 14 Figure 125H monoclonal antibody')
Claims (1)
動脈硬化症認識モノクロナル抗体。 （２）モノクロナル抗体が抗ヒト動脈硬化症抗体産生細
胞とミエローマ細胞との融合細胞からのモノクロナル抗
体である請求項第１項記載のヒト動脈硬化症認識モノク
ロナル抗体。 （３）ヒト動脈硬化症抗原が家族性高脂血症患者血清に
おける抗原である請求項第２項記載のヒト動脈硬化症認
識モノクロナル抗体。 （４）ヒト動脈硬化症抗原が脳梗塞、心筋梗塞患者血清
における抗原である請求項第２項記載のヒト動脈硬化症
認識モノクロナル抗体。 （５）ヒト動脈硬化症抗原が動脈硬化病巣からの抗原で
ある請求項第２項記載のヒト動脈硬化症認識モノクロナ
ル抗体。 （６）動脈硬化病巣が内膜肥厚部分である請求項第５項
に記載のヒト動脈硬化症認識モノクロナル抗体。 （７）モノクロナル抗体が免疫グロブリンクラスＩｇＧ
である請求項第１項記載のヒト動脈硬化症認識モノクロ
ナル抗体。 （８）モノクロナル抗体が免疫グロブリンクラスＩｇＭ
である請求項第１項記載のヒト動脈硬化症認識モノクロ
ナル抗体。 （９）モノクロナル抗体が１３１Ｂまたは１２５Ｈであ
る請求項第７項または第８項のモノクロナル抗体。 （１０）ヒト動脈硬化症関連抗原を含有する溶液で、ヒ
トを除く哺乳動物を免疫し、該動物の抗体産生リンパ球
とミエローマ細胞とを細胞融合させて抗ヒト動脈硬化症
抗体産生融合細胞を単離し、次いで抗ヒト動脈硬化症抗
体産生融合細胞を培養することを特徴とするヒト動脈硬
化症認識モノクロナル抗体の製造法。 （１１）ヒト動脈硬化症関連抗原を含有する溶液が家族
性高脂血症患者血清である請求項第１０項記載のヒト動
脈硬化症認識モノクロナル抗体の製造法。 （１２）ヒト動脈硬化症関連抗原を含有する溶液が脳梗
塞、心筋梗塞患者血清である請求項第１０項記載のヒト
動脈硬化症認識モノクロナル抗体の製造法。 （１３）ヒト動脈硬化症関連抗原を含有する溶液が動脈
硬化病巣のホモゲネートである請求項第１０項記載のヒ
ト動脈硬化症認識モノクロナル抗体の製造法。 （１４）動脈硬化病巣が内膜肥厚部分である請求項第１
３項記載のヒト動脈硬化症認識モノクロナル抗体の製造
法。 （１５）モノクロナル抗体が免疫グロブリンクラスＩｇ
Ｇである請求項第１０項記載のヒト動脈硬化症認識モノ
クロナル抗体の製造法。 （１６）モノクロナル抗体が免疫グロブリンクラスＩｇ
Ｍである請求項第１０項記載のヒト動脈硬化症認識モノ
クロナル抗体の製造法。 （１７）モノクロナル抗体が１３１Ｂまたは１２５Ｈで
ある請求項第１０項のヒト動脈硬化症認識モノクロナル
抗体の製造法。 （１８）酵素または放射性同位元素で標識されたヒト動
脈硬化症認識モノクロナル抗体。（１９）放射性同位元
素が＾１＾２＾５Ｉである請求項第１８項の標識モノク
ロナル抗体。 （２０）酵素または放射性同位元素で標識されたヒト動
脈硬化症認識モノクロナル抗体からなるヒト動脈硬化症
診断用試薬。 （２１）放射性同位元素で標識されたヒト動脈硬化症認
識モノクロナル抗体からなるヒト動脈硬化症画像診断用
試薬。 （２２）ヒト動脈硬化症が心筋梗塞または脳梗塞である
請求項第２０項または２１項の診断用試薬。 （２３）放射性同位元素が＾１＾２＾３Ｉ、＾１＾２＾
５Ｉ、又は＾１＾３＾１Ｉである請求項第２２項の診断
用試薬。 （２４）放射性同位元素が＾１＾１＾１Ｉｎである請求
項第２２項の診断用試薬。 （２５）放射性同位元素が＾９＾９＾ｍＴｃである請求
項第２２項の診断用試薬。[Scope of Claims] (1) A human arteriosclerosis-recognizing monoclonal antibody that specifically recognizes a human arteriosclerosis-related antigen. (2) The monoclonal antibody recognizing human arteriosclerosis according to claim 1, wherein the monoclonal antibody is a monoclonal antibody derived from a fused cell of an anti-human arteriosclerosis antibody-producing cell and a myeloma cell. (3) The monoclonal antibody recognizing human arteriosclerosis according to claim 2, wherein the human arteriosclerosis antigen is an antigen in serum of a patient with familial hyperlipidemia. (4) The monoclonal antibody recognizing human arteriosclerosis according to claim 2, wherein the human arteriosclerosis antigen is an antigen in the serum of patients with cerebral infarction or myocardial infarction. (5) The monoclonal antibody recognizing human arteriosclerosis according to claim 2, wherein the human arteriosclerosis antigen is an antigen from an arteriosclerosis lesion. (6) The human arteriosclerosis-recognizing monoclonal antibody according to claim 5, wherein the arteriosclerotic lesion is an intimal thickened area. (7) The monoclonal antibody is an immunoglobulin class IgG
The human arteriosclerosis-recognizing monoclonal antibody according to claim 1. (8) Monoclonal antibody is immunoglobulin class IgM
The human arteriosclerosis-recognizing monoclonal antibody according to claim 1. (9) The monoclonal antibody according to claim 7 or 8, wherein the monoclonal antibody is 131B or 125H. (10) Immunize a mammal other than a human with a solution containing a human arteriosclerosis-related antigen, and fuse the animal's antibody-producing lymphocytes and myeloma cells to produce anti-human arteriosclerosis antibody-producing fused cells. 1. A method for producing a monoclonal antibody recognizing human arteriosclerosis, which comprises isolating and then culturing a fused cell that produces an anti-human arteriosclerosis antibody. (11) The method for producing a monoclonal antibody recognizing human arteriosclerosis according to claim 10, wherein the solution containing the human arteriosclerosis-related antigen is familial hyperlipidemia patient serum. (12) The method for producing a human arteriosclerosis-recognizing monoclonal antibody according to claim 10, wherein the solution containing the human arteriosclerosis-related antigen is serum from patients with cerebral infarction or myocardial infarction. (13) The method for producing a monoclonal antibody recognizing human arteriosclerosis according to claim 10, wherein the solution containing the human arteriosclerosis-related antigen is a homogenate of arteriosclerosis lesions. (14) Claim 1, wherein the arteriosclerotic lesion is an intimal thickened area.
The method for producing a monoclonal antibody recognizing human arteriosclerosis according to item 3. (15) Monoclonal antibody is immunoglobulin class Ig
11. The method for producing a monoclonal antibody recognizing human arteriosclerosis according to claim 10, wherein the monoclonal antibody recognizes human arteriosclerosis. (16) Monoclonal antibodies are immunoglobulin class Ig
11. The method for producing a human arteriosclerosis-recognizing monoclonal antibody according to claim 10, wherein the antibody is M. (17) The method for producing a human arteriosclerosis-recognizing monoclonal antibody according to claim 10, wherein the monoclonal antibody is 131B or 125H. (18) Enzyme- or radioisotope-labeled human arteriosclerosis-recognizing monoclonal antibody. (19) The labeled monoclonal antibody according to claim 18, wherein the radioactive isotope is ^1^2^5I. (20) A human arteriosclerosis diagnostic reagent comprising a human arteriosclerosis-recognizing monoclonal antibody labeled with an enzyme or a radioactive isotope. (21) A human arteriosclerosis imaging diagnostic reagent comprising a radioisotope-labeled human arteriosclerosis-recognizing monoclonal antibody. (22) The diagnostic reagent according to claim 20 or 21, wherein the human arteriosclerosis is myocardial infarction or cerebral infarction. (23) Radioactive isotopes are ^1^2^3I, ^1^2^
23. The diagnostic reagent according to claim 22, which is 5I or ^1^3^1I. (24) The diagnostic reagent according to claim 22, wherein the radioactive isotope is ^1^1^1In. (25) The diagnostic reagent according to claim 22, wherein the radioactive isotope is ^9^9^mTc.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000558160A CA1320461C (en) | 1988-02-04 | 1988-02-04 | Monoclonal antibody capable of recognizing human arteriosclerosis and process for preparing same |
| JP63050162A JP2818658B2 (en) | 1988-03-03 | 1988-03-03 | Monoclonal antibody recognizing human atherosclerosis and method for producing the same |
| AT89103754T ATE100149T1 (en) | 1988-03-03 | 1989-03-03 | MONOCLONAL ANTIBODY CAPABLE OF RECOGNIZING AN ANTIGEN ASSOCIATED WITH HUMAN ARTERIOSCLEROSIS AND METHODS FOR ITS PRODUCTION. |
| EP89103754A EP0334076B1 (en) | 1988-02-04 | 1989-03-03 | Monoclonal antibody capable of recognizing an antigen associated with human arteriosclerosis, and process for preparing the same |
| ES89103754T ES2050171T3 (en) | 1988-02-04 | 1989-03-03 | MONOCLONAL ANTIBODY ABLE TO RECOGNIZE HUMAN ARTERIOSCLEROSIS AND PROCESS TO PREPARE IT. |
| DE68912165T DE68912165T2 (en) | 1988-02-04 | 1989-03-03 | Monoclonal antibody capable of recognizing an antigen associated with human arteriosclerosis and process for its preparation. |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63050162A JP2818658B2 (en) | 1988-03-03 | 1988-03-03 | Monoclonal antibody recognizing human atherosclerosis and method for producing the same |
| CA 588160 CA1337064C (en) | 1989-01-13 | 1989-01-13 | Draw bar sprayer kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01224400A true JPH01224400A (en) | 1989-09-07 |
| JP2818658B2 JP2818658B2 (en) | 1998-10-30 |
Family
ID=25672375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63050162A Expired - Lifetime JP2818658B2 (en) | 1988-02-04 | 1988-03-03 | Monoclonal antibody recognizing human atherosclerosis and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2818658B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07238098A (en) * | 1994-02-24 | 1995-09-12 | Betsuseru Res Lab:Kk | Monoclonal antibody for recognizing antigen related to human pultaceous sclerogenous focus |
| JP2006519032A (en) * | 2002-12-02 | 2006-08-24 | エダ リサーチ アンド ディベロップメント カンパニー リミティド | Characterization of arteriosclerosis by optical imaging |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009154026A1 (en) * | 2008-06-20 | 2009-12-23 | 国立大学法人岡山大学 | Antibody against calcified globule and use of the same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61130238A (en) * | 1984-11-30 | 1986-06-18 | Res Dev Corp Of Japan | Monoclonal antibody for recognizing arteriosclerosis focus and reagent |
-
1988
- 1988-03-03 JP JP63050162A patent/JP2818658B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61130238A (en) * | 1984-11-30 | 1986-06-18 | Res Dev Corp Of Japan | Monoclonal antibody for recognizing arteriosclerosis focus and reagent |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07238098A (en) * | 1994-02-24 | 1995-09-12 | Betsuseru Res Lab:Kk | Monoclonal antibody for recognizing antigen related to human pultaceous sclerogenous focus |
| JP2006519032A (en) * | 2002-12-02 | 2006-08-24 | エダ リサーチ アンド ディベロップメント カンパニー リミティド | Characterization of arteriosclerosis by optical imaging |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2818658B2 (en) | 1998-10-30 |
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