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JPS6134097B2 - - Google Patents
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JPS6134097B2 - - Google Patents

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Publication number
JPS6134097B2
JPS6134097B2 JP58165330A JP16533083A JPS6134097B2 JP S6134097 B2 JPS6134097 B2 JP S6134097B2 JP 58165330 A JP58165330 A JP 58165330A JP 16533083 A JP16533083 A JP 16533083A JP S6134097 B2 JPS6134097 B2 JP S6134097B2
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JP
Japan
Prior art keywords
antigen
solution
antibody
capillary
molar
Prior art date
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Expired
Application number
JP58165330A
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English (en)
Other versions
JPS5990056A (ja
Inventor
Orofu Gurutsubu Anderusu
Kurisuchina Guratsudo Uura
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Syva Co
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Syva Co
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Publication of JPS5990056A publication Critical patent/JPS5990056A/ja
Publication of JPS6134097B2 publication Critical patent/JPS6134097B2/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/97—Test strip or test slide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/80—Fluorescent dyes, e.g. rhodamine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

【発明の詳細な説明】 技術分野 本発明は免疫化学的定量方法に関し、更に詳し
くは毛細管作用をなす多孔質担体物質を用いた免
疫化学的定量方法に関する。
発明の目的及び構成 本発明は臨床検査等において目的とする検査を
免疫化学的定量により実施するのに際し、所望の
免疫化学的定量を迅速、簡便かつ安定にしかも高
精度で行なうことができる免疫化学的定量方法を
提供することを目的とする。
本発明に従えば、抗体を結合せしめた毛細管含
有多孔質担体物質から成る試験片を定量すべき抗
原を含む試料と接触させて免疫化学的に定量する
方法において、 (i) 抗原の毛細管移行を試験片中において開始せ
しめ、 (ii) 該試験片を呈色指示薬と結合した抗体で処理
して試料中の抗原含量の増加と共にその度合が
増大する、試験片中の抗原で覆われた部分を呈
色せしめ、そして (iii) 抗原の移行距離を測定することによつて試料
中の抗原含量を定量することを特徴とする免疫
化学的定量方法が提供される。
発明の具体的説明 本発明に従つた免疫化学的定量方法の抗体を結
合せしめた多孔質担体物質は、好ましくは、ポリ
塩化ビニル、塩化ビニル−プロピレン共重合体又
は塩化ビニル−酢酸ビニル共重合体のマトリツク
ス中に実質上均一に埋封されたシリカ微粉から成
る。
本発明の臨床検査材は、抗体を結合させた毛細
管の作用をなす多孔質担体から成り、例えば吸着
または臭化シアンあるいはグルタールアルデヒド
のような物質により公知の方法により共有結合で
抗体に結合させた多孔質の毛細管の作用を利用す
ることを特徴とするものである。
即ち、本発明に従えば、定量しようとする抗原
を含む試料を、毛細管の作用をなさしめる条片に
浸透せしめたのち、条片の抗原を含有する部分
は、螢光化合物又は発色反応を触媒する酵素のよ
うな適当な呈色指示薬に結合された抗体を添加す
ることにより顕色させて検出するものである。
従来の免疫化学的定量法は、例えばマンシーニ
法やオーチヤロニー法のような免疫拡散法あるい
はローレルの免疫電気泳動法によるものである
が、これらの方法においては、その変法も含めて
拡散や電気泳動過程によつて抗原と抗体との相互
作用を必要とするが、本発明の場合は、抗体を結
合させた多孔質担体物質の毛細管の作用によつて
抗原と抗体の相互をなさしめるものである。
本発明の実施のための毛細管作用をする物質と
しては、紙、クロマトグラフ用紙、イオン交
換紙、セルロースアセテート膜、セルロースアセ
テートデイスク、セルロース薄膜クロマトグラフ
用デイスク等のようなセルロース繊維を含む物質
や、デンプン、セフアデツクス（三次元構造の網
状組織又はエピクロルヒドリンと交さ結合したデ
キストラン鎖のマトリツクス、フアルマシアフア
インケミカルス社、ウプサラ、スウエーデン製）
のような物質や、ポリ塩化ビニル、塩化ビニル−
プロピレン共重合体、塩化ビニル−酢酸ビニル共
重合体のようなプラスチツク物質、セラミツク物
質およびポリ塩化ビニル・シリカを結合させたも
のが利用できる。また本発明において用いられる
抗体は、公知の免疫法に用いられるものである。
実施例 以下に本発明の実施例を示す。
臨床検査材の製法 例 １ 毛細管物質における臭化シアンによる抗体の不
溶化 セルロース含有物質10ｇを、蒸留水に５分間浸
し、これに300mlの蒸留水に臭化シアン８ｇを溶
かした溶液を加え、１規定苛性ソーダでPHを10.5
に調整する。このPH値に保つことにより反応は20
分間で完結するから、溶液を捨て、更にセルロー
ス物質を２の0.005モル炭酸水素ナトリウムで
洗浄し、洗浄液を除去したのち、このセルロース
物質を、0.1モル炭酸水素ナトリウム10mlに500mg
の抗ヒドガンマグロブリン（IgG.Fc分画）家兎
ガンマグロブリン含有溶液中４℃で１夜振とう
し、更に0.005モルエタノールアミン溶液100mlを
用いて３時間振とうし、しかる後に0.5モル炭酸
水素ナトリウム500ml、次に、PH4.0の0.1モル酢
酸緩衝液200mlで洗浄して、0.3％アルブミンを加
えた0.075モルバルビツール酸ナトリウム緩衝液
500ml中に保存する。この保存温度は４℃とす
る。
例 ２ 毛細管物質におけるグルタールアルデビドによ
る抗体の不溶化 セルロースアセテート膜0.6ｇを、PH6.8の0.1モ
ルリン酸緩衝液10ml中に抗ヒトガンマグロブリン
（IgG.Fc分画）家兎ガンマグロブリン100mgを溶
解した溶液中で30分間振とうし、その溶液を捨て
たのち、膜を１％グルタールアルデヒド溶液10ml
中で更に30分間振とうし、ついで1.0モルメチル
アミン10ml中で15分間振とうする。しかる後に膜
を100mlのバルビツール酸ナトリウムで洗浄し、
これを同じ緩衝液に、0.3％アルブミンを加え
て、その溶液中において４℃で保存する。
例 ３ 毛細管担体における吸着による抗体の不溶化 ポリ塩化ビニル・シリカ（ミクロポーラスプラ
スチツクシート・アメリカエスナコーポレーシヨ
ン社製）10ｇを、PH6.8の0.1モルリン酸緩衝液
100mlに、抗ヒトガンマグロブリン（IgG.Fc分
画）家兎ガンマグロブリン500mgを含む溶液中に
おいて４℃で１夜振とうし、、完全に反応させた
のち、目的物をPH7.0にリン酸で緩衝化された生
理食塩液１を用いて洗浄し、これを紙に挾ん
で乾燥させる。
例 ４ 螢光標識抗体の製法 (a) 抗ヒトガンマグロブリン（IgG、Fc分画）家
兎ガンマグロブリン720mg、塩化ナトリウム
0.57ｇ、炭酸水素ナトリウム0.259ｇおよび炭
酸ナトリウム・10水和物0.045ｇを蒸留水72ml
に溶解し、反応溶液を氷浴中で冷却し、撹拌し
ながらフロレツセインイソチオシアネート36mg
を加え、さらに溶液を撹拌しながら18時間冷却
する。その混合物を透析溶液が螢光を失うま
で、リン酸で緩衝化されたPH7.0の生理食塩液
に対して透析し、得られた標識抗体を凍結して
保存する。
(b) 抗ヒトガンマグロブリン（IgG.Fc分画）家
兎ガンマグロブリン720mg、塩化ナトリウム
0.57ｇ、炭酸水素ナトリウム0.259ｇおよび炭
酸ナトリウム・10水和物0.049ｇを72mlの蒸留
水に溶解し、反応溶液を氷浴で冷却し、これを
撹拌しながら、ロダミン９mgを溶解したアセト
ン２mlをこれに加え、その溶液を18時間冷却す
る。これを透析溶液が螢光を失うまで、リン酸
でPH7.0に緩衝化された生理食塩液に対して透
析する。このようにして得られた標識抗体を凍
結して保存する。
(c) 抗ヒトガンマグロブリン（IgG.Fc分画）家
兎ガンマグロブリン720mg、塩化ナトリウム
0.57ｇ、炭酸水素ナトリウム0.259ｇおよび炭
酸ナトリウム・10水和物0.049ｇを、72mlの蒸
留水に溶解し、その反応液を氷浴で冷却し、こ
れを撹拌しつゝ18時間冷却し、しかる後に、そ
の混合物を、リン酸で緩衝化されたPH7.0の生
理食塩液に対して透析液の螢光がなくなる迄、
透析する。斯して得られた標識抗体を凍結保存
する。
例 ５ LDHアイソ酵素H4標識抗体の製法 硫酸アンモニウムで沈澱させたLDHアイソ酵
素H44.5mgの懸濁液を、PH6.8の0.1モルリン酸緩
衝液で透析し、撹拌しつゝ内液に抗ヒトガンマグ
ロブリン（IgG、Fc分画）家兎ガンマグロブリン
2.25mgを加えて、リン酸緩衝液で最終量を1.35ml
とし、全ての家兎ガンマグロブリンが溶解したの
ち、これに0.1％グルタールアルデヒド溶液45μ
ｌを滴下し、その反応液を室温中で２時間放置
し、更にその混合物をバルビツール酸ナトリウム
緩衝液に対して透析し、得られた標識体を４℃に
おいて保存する。而してこれを使用する場合に
は、２％アルブミンを加えたバルビツール酸緩衝
液で希釈するのである。
例 ６ 不溶化の点検 前述の例に従つて抗体と処理した物質の２つの
条片と、未処理の２つの条片とを試験に用いる。
即ち、抗体処理条片１つおよび未処理条片１つ
を、バルビツール酸緩衝液１ml当りヒトガンマグ
ロブリン（IgG）１mgの溶液に浸漬する。つぎに
４つの条片を0.15モル塩化ナトリウムを含むバル
ビツール酸緩衝液20mlにより10分間洗浄したの
ち、更にそれらをフロレツセインイソチオシアネ
ート標識抗体中で10分間振とうする。洗浄をくり
返したのち、条片を乾燥し、340nｍの波長の紫
外線で検査し、IgG溶液で振とうした不溶化抗体
を含浸した条片にのみ螢光が現われることを確認
する。例３の方法により製造した条片を検査する
場合には、ウシアルブミンで処理した条片を対照
として用いる。
免疫化学的定量法 例 ７ 本発明によつて製造した臨床検査材を、免疫化
学的定量として診断の目的のために用いる場合に
は次の方法による。
即ち、１〜３μｌの既知容量の被検物質と、既
知量の抗原を含む相応量の標準溶液およびそれを
種々希釈したものを、同じく担体物質上に付着す
る。この場合、移動相が毛細管内を移行し易いよ
うに、例えば0.3％アルブミンを添加した0.075モ
ルバルビツール酸ナトリウム緩衝液を用い、毛細
管による滲透作用は、開放状態または密閉試験容
器中で行わせる。この滲透は上向または下向で差
支えないが、特に密閉容器と上向浸透の組み合わ
せが望ましい。毛細管の浸透が開始した後の適当
な時期に、湿つた担体物質を、フロレツセインイ
ソチオシアネートまたは乳酸デヒドロゲナーゼ
（LDH）標識抗体を含む溶液に移すのであるが、
この溶液中での振とう時間は10分程度が望まし
い。振とう後、担体物質を37℃の流水で５分間程
度洗浄し、更に0.2％アルブミンと0.15モル塩化
ナトリウムを含む20mlのバルビツール酸ナトリウ
ム緩衝液で５分間ずつ２回振とうする。
抗体の移動距離を測定するために、螢光抗体が
用いられるが、その観察を340nｍの紫外線で行
う。若し、LDH抗体溶液が用いられる場合に
は、担体物質は下記の組成の染色浴で処理する。
即ち、ニトロブルーテトラゾリウム30mgと
NAD40mg、PH7.4の0.05モルトリス緩衝液56mlに
溶解し、この液に3.6モル乳酸リチウム1.60ml、
0.06モルシアン化カルシウム５mlおよび数粒のメ
チルフアナゾリウムメトサルフエートを、２mlの
蒸留水に溶解した溶液を加え、37℃の染色浴中
で、毛細管物質に明瞭な色彩が観察されるまで振
とうする。色彩の顕色は、乳酸＋NADピルビ
ン酸＋NADH2の反応に、LDHが触媒として作用
し、NADH2が定量的にニトロブルーテトラゾリ
ウムを還元して、不溶性の淡紫色々素を作るため
である。
臨床検査材の抗原で被覆された部分の呈色は、
被検物質の抗原の含有量が増すにつれて強度を増
す。格子に結合された抗体分子は、毛細管による
浸透中に交換反応によつて抗原の移動を遅延させ
る。抗原の濃度が高くなるに従つて、抗体に結合
されている抗原分子の平均移動時間が減少するた
めに、遅延効果が少なくなるのである。
例 ８ 例えば本発明によつて製造された毛細管作用を
なす抗体結合担体物質の条片を、被検物質の少量
の溶液に３mmの深さに浸し、毛細管の浸透を開始
させて３〜20分間続けた後、条片を溶液から引上
げて、抗原の移動距離を例７において述べた要領
で測定する。
こゝには一部の実施例について説明したが、こ
実施例は種々の等価な変更や置換が可能である。
本発明において使用する臨床検査材は多孔性
（即ち、毛細管の作用をなす）担体物質の条片よ
りなる、「条片」という用語は厚味のあるろ紙
や、膜状又は円板状、更には薄い平たい棒状のも
の等を広義に含むものである。
「担体」という用語は抗体に対し例えば吸着或
いは共有結合で結合しうる物質を意味するもの
で、前述の物質に限るものではない。
例３の多孔性ポリマーマトリツクスに配置され
た均一に細分化されたシリカを有する塩化ビニー
ルのミクロポーラスプラスチツクシートは他の適
当な浸透性又は多孔性の、同様にシリカ微粉が埋
封された塩化ビニル−プロピレン共重合体或いは
塩化ビニル−酢酸ビニル共重合体等の連続する重
合マトリツクスに代えてもよい。又そのミクロポ
ーラスプラスチツクシートにおき代えられる。ミ
クロポーラスシートの孔の直径は0.1〜１ミクロ
ンである。又全体の孔の量はミクロポーラスプラ
スチツクシートの１グラムあたり1.1〜1.8ミリリ
ツトルである。

Claims (1)

  1. 【特許請求の範囲】 １ 抗体を結合せしめた毛細管含有多孔質担体物
    質から成る試験片を定量すべき抗原を含む試料と
    接触させて免疫化学的に定量する方法において、 (i) 抗原の毛細管移行を試験片中において開始せ
    しめ、 (ii) 該試験片を呈色指示薬と結合した抗体で処理
    して試料中の抗原含量の増加と共にその度合が
    増大する、試験片中の抗原で覆われた部分を呈
    色せしめ、そして (iii) 抗原の移行距離を測定することによつて試料
    中の抗原含量を定量することを特徴とする免疫
    化学的定量方法。
JP58165330A 1975-01-27 1983-09-09 免疫化学的定量方法 Granted JPS5990056A (ja)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE75008417 1975-01-27
SE7500841A SE388694B (sv) 1975-01-27 1975-01-27 Sett att pavisa ett antigen exv i prov av kroppvetskor, med utnyttjande av till porost berarmaterial bundna eller adsorberande antikroppar

Publications (2)

Publication Number Publication Date
JPS5990056A JPS5990056A (ja) 1984-05-24
JPS6134097B2 true JPS6134097B2 (ja) 1986-08-06

Family

ID=20323501

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Application Number Title Priority Date Filing Date
JP51007875A Pending JPS51101122A (ja) 1975-01-27 1976-01-27
JP58165330A Granted JPS5990056A (ja) 1975-01-27 1983-09-09 免疫化学的定量方法

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Application Number Title Priority Date Filing Date
JP51007875A Pending JPS51101122A (ja) 1975-01-27 1976-01-27

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US (1) US4168146A (ja)
JP (2) JPS51101122A (ja)
AT (1) AT347601B (ja)
AU (1) AU508642B2 (ja)
BE (1) BE837896A (ja)
CA (1) CA1074228A (ja)
DE (1) DE2603004C2 (ja)
DK (1) DK149968C (ja)
FI (1) FI760074A7 (ja)
FR (1) FR2298798A1 (ja)
GB (1) GB1502563A (ja)
IL (1) IL48722A (ja)
IT (1) IT1123602B (ja)
NL (1) NL7600128A (ja)
NO (1) NO760233L (ja)
NZ (1) NZ179830A (ja)
SE (1) SE388694B (ja)
ZA (1) ZA76421B (ja)

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IL48722A0 (en) 1976-02-29
SE388694B (sv) 1976-10-11
AU508642B2 (en) 1980-03-27
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NL7600128A (nl) 1976-07-29
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NO760233L (ja) 1976-07-28
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US4168146A (en) 1979-09-18
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DE2603004A1 (de) 1976-07-29
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IT1123602B (it) 1986-04-30
FR2298798A1 (fr) 1976-08-20
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AU1056376A (en) 1977-07-28

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