For the rapid and sensitive analysis of proteins, and to obtain reproducible results, an apparatus for isotachophoresis, Shimadzu IP-2A, was modified and the electrophoretic conditions were standardized. The apparatus was modified as follows; 1) Two peristaltic pumps were used instead of N₂ gas pressure system, for rinsing and refilling the capillary tube and the electrode chambers. 2) The flow lines were simplified into 3 lines, instead of 10 lines of the commercial apparatus. The electrophoretic conditions were changed as follows; 1) Hexane was overlayed on the electrolyte solutions to minimize the solubilization of CO₂, instead of the use of Ba(OH)₂. 2) The inner surface of the capillary tube was coated with hydroxypropyl methyl cellulose (HPMC), instead of using HPMC-containing leading electrolyte. The amount of the ampholyte mixture (Ampholine pH 3.5-10) to be applied was standardized to be 20μg. From these improvements and standardization, the interruption of electrophoresis by N₂ gas bubbles or by the precipitation of BaCO₃ were avoided and the procedure of isotachophoretic analysis was simplified. Serum proteins as low as 3μg could be separated into 20 UV-absorbing peaks within 16min of running time in good reproducibility.

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In order to evaluate the improved technique of capillary isotachophoresis, human sera from 10 normal subjects and 22 patients of monoclonal gammopathy (IgG type) were analyzed. The value of potential gradient was used to represent the mobility of each UV absorbing peak. All the sera from the patients were characterized by one or a series of sharp UV absorbing peak, which was at least 3-fold higher than the corresponding IgG peak of normal subject. Comparing the results with those obtained by polyacrylamide micro two-dimensional electrophoresis, it was as certained that the value of potential gradient of a monoclonal IgG molecule is related to its isoelectric point. Because of the highly simplified operation and the high resolution of proteins, the technique is promising for the routine analysis of serum proteins in clinical laboratories.

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This report present data on the genetic polymorphisms of human serum transferrin in the Japanese population have been described using capillary type isotachophoresis. Tf phenotypes Tf BC, Tf C and Tf CD were detected. In randam samples of 70 and 112 unrelated healthy individuals from the Kohama Island of Okinawa Prefecture and the Oki Island of Shimane Prefecture, respectively.
The gene frequencies obserbed were Tf B: O (Kohama) and 0.0048 (Oki), Tf C₁: 0.6143 (Kohama) and 0.801 (Oki), Tf C₂: 0.3071 (Kohama) and 0.1748 (Oki) and Tf D: 0.0786 (Kohama) and 0.0194 (Oki). Frequencies of Tf C was compared with other reports. The gene frequencies of Tf B, C₁, C₂ and D in Tokyo population (B: 0.0026, C₁: 0.6969, C₂: 0.2979 and D: 0.0026) were higher than in Kohama Island population (0, 0.6143, 0.3071 and 0.0786) and Oki Island population (0.0048, 0.8010, 0.1748 and 0.0194).
In order to detected the serum transferrin phenotypes the capillary type isotachophoresis technique was more suitable than the polyacrylamide gel isoelectrofocusing and one dimensional electrophoresis technique.

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下剤として用いられて来ている植物性和漢生薬のダイオウ (Rheim palmatum 等の鱗茎) およびセンナ (Cassia angustiflorum 等の葉) の有効成分センノサイド類の主成分であるセンノサイドAおよびB (ジアンスロン誘導体で相互に立体異性体) の分離・分析の方法を, 細管式等速電気泳動法を用いて確立した. 実際の植物生薬抽出液中のセンノサイドA (PU値0.39) およびB (0.48) も, 標品との混合分析におけるゾーン長の延長により同定された. また, ロットの異なる合計30の生薬試料について本法によりセンノサイドAおよびBの含量を測定したところ, 得られた結果に見られたダイオウ, センナにおける両成分の含量とA/Bの量比についての傾向は, 初期の研究者による報告のものと一致した. さらに, うち10例について, 薄層クロマトグラフィー(TLC) 法による両成分の定量も行って値を比較したところ, センノサイドA(r=0.91), B(r=0.87) とも良好な相関性を示した. 以上より, 本法は, 生薬試料中のセンノサイドAおよびBを定量する迅速微量分析の系として, 在来法の一つであるTLC法と比較して少なくとも同程度には信頼して用いられるものである, と思われた.

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Computer simulation of isotachophoresis of proteins using carrier ampholytes such as Ampholine or Sepaline as the spacers was performed on the basis of the author's theory of steady state electrophoresis. As carrier ampholytes behave as if they were weak acids in anionic isotachophoresis, the simulations were performed on systems composed of five weak acids with similar pK values in the absence or in the presence of up to two protein components. The results could explain those of agarose gel isotachophoresis experiments of proteins using carrier ampholytes as the spacers.

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ヒト精漿の個人識別に有用な標識を検索することを目的として, ミクロ2次元ポリアクリルアミドゲル電気泳動法 (M2D-PAGE) および細管式等速電気泳動法(CITP) の有用性を検討した. M2D-PAGEにより精漿のAcp, Alb, Tf, γ-Smのスポットはその染色強度, 数などに個体差を見いだした. また, CITPのUV吸収のピーク面積の個体差にM2D-PAGEとの関連性を認めた. 2次元目を6～25%としてM2D-PAGEを銀染色した結果, 分子量5000付近の低分子領域に13個のスポットが検出された. そのうちの3個の低分子精漿蛋白質 (Lsp) に個体差が認められ, その変異は6種の表現型として観察された.

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Capillary type isotachophoresis (CITP) and micro two-dimentional polyacrylamide gel electrophoresis (M2D-PAGE) were used by examining the genetic polymorphisms and the physiological variation of horse serum proteins. Both methods were useful for evaluating Tf typings. Especially, the CITP was a rapid analytical method. One of the very rare Tf type, Tf DM, which was undetectable by conventinal methods, was discovered by using this method. The amount of various proteins were estimated from the data on UV absorption. The Tf heterogenity was detectable by the M2D-PAGE. In addition, this method proved to be an excellent one for observing physiological conditions of each samples.

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On the red blood cell membrane in a patient with autoimmune hemolytic anemia (AIHA) several glycophorin class blood group antigens were assumed to be acquired in relation to the disease. When the red blood cells were treated with Dispase, all of the acquired antigens were released to the supernatant in company with the native glycophorin class antigens and disappeared on the red blood cell membrane. Then a trial was made to analyze the supernatant of the red blood cell suspension with Dispase and the serum which contained the red cell autoantibody by capillary isotachophoresis. In each of the isotachopherogram, a specific peak which was not detected in the sample from normal human was found in the acidic zone. The protein substances expressed by these peaks will be available as a marker to diagnose AIHA because they are thought to be formed in association with AIHA.

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Electrophoretic pattern of human leucocyte cell extracts was surveyed by isotachophoresis.

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