Cellulose acetate membrane isoelectric focusing (CAIEF) of streptolysin O from Group A streptococci and direct detection method of hemolytic activity by red blood cell agar layering after focusing are described. Eleven hemolytic components from concentrated culture filtrate were detected which had pI values of 4.8, 5.0, 5.3, 5.4, 5.6, 5.9, 6.4, 6.6, 6.9, 7.2 and 7.4. Sensitivity of the direct detection method of hemolytic activity was examined with pI 6.5 hemolytic component prepared by preparative gel isoelectric focusing. This method was extremely sensitive, which was possible to detect 0.4 HD₅₀ of streptolysin O. These methods were applied to the analysis of the fractions obtained by preparative polyacrylamide or Sephadex gel isoelectric focusing of concentrated culture filtrate. Reproducibility of pI value in CAIEF was comparable to that in polyacrylamide gel isoelectric focusing. The method is inexpensive and enables easy detection of hemolytic toxin.

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A method for the routine classification of hyperlipidemia according to Fredrickson is described, which includes the determination of cholesterol, triglycerides, and electrophoretic analysis of the lipoproteins on cellulose acetate.
Satisfactory separation of lipoprotein fractions, particularly beta and pre-beta, improved readability and reproducibility are achieved with the electrophoresis for lipoproteins using Sepraphore III (Gelman Instrument Co.).
Plasma samples from 199 healthy, fasting subjects were analysed, and normal values of cholesterol, triglycerides, and lipoproteins on the cellulose acetate electrophorogram are presented in terms of age and sex.
The values can serve as a baseline of the most essential parameters for accurate differentiation of the types of hyperlipidemia.

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Separax 膜と反応するIgM-κ型M蛋白を見いだし検討を加えた. ジアセチルセルロースとトリアセチルセルロースを等量混合し油性成分を含まないセ・ア膜を用い電気泳動を行ったところ, 明瞭なM蛋白帯は認めず原点のα₁からβ位に残る幅広い異常蛋白帯が観察された. 水酸基の多い電気浸透除去剤であるヒドロキシプロピルセルロースを含有した緩衝液によるSeparax 膜電気泳動では, Separax-SP膜と同様, 明瞭なM蛋白帯が観察され, 従来のセ・ア膜のアセチル基を水酸基に変えたミクロフィルターFR 100でも明瞭なM蛋白帯が観察された. 患者M蛋白と Separax 膜のアセチル基との反応が考えられ, セルロース膜による血液透析などの既往歴がまったくないことから, その反応様式は免疫学的な交差反応に基づいている可能性が考えられた.

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Various different stainings were tested with cellulose acetate membrane as a supporting medium, in order to study their sensitivities for staining protein fractions at very low concentrations. The pigment Acid violet 17 was found to be most sensitive, requiring only a sample size of 1μl for a sample with a total protein concentration of 200mg/dl and 4μl for a total protein concentration of 50mg/dl. Acid violet 17 staining was approximately 10 times more sensitive than Ponceau 3R staining, and since cerebrospinal fluid may be analysed in the unconcentrated form at this sensitivity, it promises to be clinically useful. Acid violet 17 staining is easy to perform, and gave advantages over other stainings in terms of sensitivity, rapidity, and economy, hence we believe Acid violet 17 staining provides a very suitable method for routine analysis.

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Serum protein fractionation has been performed by electrophoresis on cellulose acetate membrane in clinical laboratory and has been applied to diagnostic procedure for pathological states. In Japan SEPARAX-SP (Fuji film Co.) has been used as the supporting media of cellulose acetate membrane for about twenty years and will finish its production soon. A new cellulose acetate membrane, SELECA-VSP (Toyo Roshi Kaisha Ltd.) has been developed instead of SEPARAX-SP. The SELECA-VSP has almost similar features and function. In this study we evaluated quality and utility of SELECA-VSP by automated electrophoresis system (Olympus AES320). Protein fractionation using SELECA-VSP was not worse than that of SEPARAX-SP. Separation sensitivity was higher in SELECA-VSP. However, irregularly smeared bands were sometimes observed adjacent to monoclonal protein bands. Areas of each fraction are highly correlated in between SEPARAX-SP and SELECA-VSP. The fraction area of α₁-globulin was significantly different in SEPARAX-SP and SELECA-VSP, and about 0.01 g / dl higher in SELECA-VSP. After the presence of the difference is recognized, automated classification system for pathological states by protein fractionation could be utilized for routine laboratory testing.
In conclusion, the new cellulose acetate membrane, SELECA-VSP will replace SEPARAX-SP as electrophoretic supporting media for serum protein fractionation.

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Separax-SP膜と反応するIgA₁-κ型M-蛋白を見い出し検討を加えた. このM-蛋白は, 2.0M NaClの洗浄でも吸着したセ・ア膜から解離することはなく, 還元アルキル化および neuraminidase 処理後でもセ・ア膜との反応性は消失しなかった. ジアセチルセルロースとトリアセチルセルロースを等量混合し油性成分を含まないセ・ア膜を用い電気泳動を行ったところ, 明瞭なM-蛋白帯は認めず, 原点のα₁からβ位に残る幅広い異常蛋白帯が観察され, M-蛋白とアセチルセルロースとの結合による異常が示唆された. 患者IgA₁型M-蛋白をプロテアーゼ処理後, 精製したαFab分画, およびαFc分画単独ではセ・ア膜との反応性は消失した. これらの結果から, セ・ア膜との反応は抗原抗体反応によるものではなく, 患者IgA₁型M-蛋白がもつ物理的立体構造に起因した結合の可能性が高い.

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As a cellulose acetate membrane for serum protein fraction test, SELECA-VSP has been developed to substitute for SEPARAX-SP. Then we investigated whether SELECA-VSP can be used for protein fraction test by use of full-automated electrophoresis system (JOKOH CTE series).
A difference was observed in the A / D sensor level detecting that SELECA-VSP fed into buffer bath because SELECA-VSP was thinner than SEPARAX-SP. Therefore, the adjustment of the A / D sensor level is possibly required when SELECA-VSP is substituted for SEPARAX-SP.
On the other hand, there were no difference in membrane feeding test, electrophoretic properties and measured percentage of each protein fraction of control serum. Slight change in the full-automated electrophoresis system parameter easily leads to apply SELECA-VSP to routine laboratory test using JOKOH CTE series electrophoresis system.

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SEPARAX-SP membrane is used generally as supporting medium for serum protein fractionation in many clinical laboratories, but it has not been used for isoelectric focusing (IEF). Although the exclusive cellulose acetate membrane for IEF is a SEPARAX-EF membrane, we studied usefulness of SEPARAX-SP membrane as supporting medium for cellulose acetate membrane IEF as comparison with SEPARAX-EF membrane. Serum sample was applied and electrophoresed in according to our method. After electrophoresis, all cellulose acetate membranes were stained with Coomassie brilliant blue G-250. The both membranes showed clear and similar isoelectrophoretic patterns of serum protein. Furthermore, we confirmed that isoelectric focusing can be carried out on six layers of SEPARAX-SP membranes as same as SEPARAX-EF membrane. All six SEPARAX-SP membranes were showed clear isoelectrophoretic patterns of serum protein, but detected serum protein concentration was decreased in lower layers of SEPARAX-SP membrane. Therefore, we examined the distribution of protein among the six layers of SEPARAX-SP membrane and compared to those of SEPARAX-EF membrane. The ratio of protein distribution between top membrane and bottom membrane was 2:1 on SEPARAX-SP membrane against to the ratio of 30:1 on SEPARAX-EF membrane. Furthermore, protein distribution of six layers on SEPARAX-SP membrane was more uniform than that on SEPARAX-EF membrane. These results indicated that SEPARAX-SP membrane was useful supporting medium for cellulose acetate membrane IEF.

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Two types of new cellulose acetate membrane, Seleca-V membrane, were developed; G membrane with mild electro osmosis and A membrane with marked electro osmosis. G membrane had its application point at the post-γ zone, and A membrane at the pre-albumin zone. G membrane was found to be supericr to A membrane in its resolution, precision and reproducibility for clinical application.

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We present here a rapid method for determining substrate specificities of protease activities by using two-dimensional polyacrylamide gel electrophoresis and cellulose acetate membranes. Proteolytic active exzymes were satisfactorily separated by the two-dimensional polyacrylamide gel electrophoresis. Determining of the substrate specificities of protease activities were accomplished by using cellulose acetate membranes as absorbent of substrates. Two sheets of cellulose acetate membranes containing different substrates respectively were placed on the both sides of the thin gel layer. During incubation, enzyme activities were transferred to the membranes from the gel layer. After incubation, the membranes were stripped from the gel layer and each substrate hydrolyzing activity was detected. Comparing the zymograms, substrate specificities of proteolytic active spots were easily determined. This method is sensitive and rapid, allowing us to determine the precise substrate specificities of protease activities from only one gel layer.

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New cellulose acetate membrane SELECA-VSP was prepared as a substitute for the conventional cellulose acetate membrane SEPARAX-SP, which has been widely used for serum protein electrophoretic fractionation. We evaluated the separating quality of this new membrane, using fully automated serum protein electrophoresis system AES630.
Correlation of serum protein fraction values (percentage composition) between the two membranes, those patterns of sera from patients with protein disorders, and reference intervals in healthy adults were analyzed. In addition, the electrophoretic determinations of amylases and glycoproteins were tested.
The correlation coefficients of the each electrophoretic fraction between SELECA-VSP and of SEPARAX-SP were greater than 0.97. Reference intervals in healthy adults on SELECA-VSP and on SEPARAX-SP were nearly equal.
The separating performance of SELECA-VSP were rather higher as compared to SEPARAX-SP for serum protein electrophoretic fractionation, amylase isoenzymes and glycoproteins.
The utility and effectiveness of a new membrane SELECA-VSP can be best appreciated on the basis of our experimental results.

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Immunostaining method for the sensitive detection of isoferritins separated by isoelectric focusing on cellulose acetate membrane is described in this paper. Methanol was used in order to fix proteins to the membrane. Membrane was shaked in methanol immediately after isoelectric focusing and then incubated in phosphate buffered saline containing a sufficiency of antibody and 2% skim milk. Blocking, a step for filling in the blank of membrane was not necessary. Fine isoferritin profile with clear background was obtained using this procedure. The sensitivity of immunostaining was slightly higher than that of gold staining in which proteins were fixed with sulfosalicylic acid and/or trichloroacetic acid. When 250ng of liver ferritin was applied on the membrane, minor acidic isoferritin bands also were observed distinctly.

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