In order to substitute SELECA-VSP for SEPARAX-SP, we investigated membrane performance difference among SELECA-VSP lots and among laboratories. We examined three lots of SELECA-VSP, namely T10, T12 and T14. Automated electrophoresis system for protein fraction test used are Olympus AES series in two laboratories and JOKOH CTE series in other two laboratories. The 4 laboratories assayed common serum samples as usual procedure of each laboratory.Among different membrane lots, good correlation between each two lot was observed in the five serum protein fractions. There was statistically significant difference among the membrane lots, however, it might be only due to good reproducibility and statistics. The bias was below clinical permission limit.Among laboratories, good correlation between each two laboratory was observed in the five serum protein fractions. The bias between laboratories is below permission limit, compared with bias detected from external quality assessment program for SEPARAX-SP.In conclusion, because the bias in membrane lots and laboratories can be ignored for laboratory diagnosis of serum protein fraction test, SELECA-VSP will come onto the market to make up for SEPARAX-SP.
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.
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.
As a cellulose acetate membrane for serum protein fractionation testing, SELECA-VSP has been developed to substitute for SEPARAX-SP. In order to investigate whether or not SELECA-VSP is available for routine laboratory testing, we evaluated basic performance of SELECA-VSP by use of automated electrophoresis system, Olympus AES series.As for the consecutive transportability, no jam was occurred on 300 consecutive membrane sheets of SELECA-VSP, lot T13-2. Reproducibility was good in within-run and between-run precision. However, the waiting coefficient for application should be set up adequately in each laboratory because SELECA-VSP tends not to dry up. Using SELECA-VSP the decolored clear zone between β globulin fraction and γ globulin fraction is more distinct than SEPARAX-SP, the results analyzed by diagnosis support program may be misinterpreted. In order to avoid the misinterpretation, the standard densitogram of normal control by use of SELECA-VSP should be prepared.
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.
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.
-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
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.
各種の胆汁酸, 抱合胆汁酸をセルロースアセテート膜, アガロースゲル膜を支持体として電気泳動を行い, 3α-ヒドロキシステロイドデヒドロゲナーゼ/ジアホラーゼ/ジホルマザン系で染色することに成功した. 胆汁の胆汁酸を同方法で分析したところ, コール酸群とケノデオキシコール酸 (あるいはデオキシコール酸) 群を識別できた. リトコール酸は検出しなかった.
健常人および患者血清タンパク質を未処理フューズドシリカキャピラリーを用いたキャピラリー電気泳動法で分離分析した. 血清タンパク質をアルブミン, α1
-グロブリン, β-グロブリン, γ-グロブリンの各分画として, それらの成分比をセルロースアセテート膜を担体とした電気泳動法より得られる結果と比較した. CEにおいて, α1
分画が多めに (CA法における相対成分比の平均値2.3に対してCE法は4.6) 検出されたことを除けば, よく一致する値を得た.
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.
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.
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.
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.