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.
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.
Following the discontinuation of manufacturing SEPARAX-SP, a cellulose acetate membrane from Fujifilm which had long been used in clinical laboratory testing, Advantec Toyo decided to manufacture another cellulose acetate membrane that exhibits the properties of SEPARAX-SP. However, it is unclear whether SELECA-VSP, the new product based on certain changes in the manufacturing process of the cellulose acetate membrane, is equivalent to SEPARAX-SP in quality. Thus, we conducted a basic study using a prototype membrane of SELECA-VSP on an automatic electrophoresis apparatus, and found that it could be applied to the currently used model of automatic electrophoresis apparatus without adjusting operating conditions. The basic study results and their correlations were promising enough to suggest that SELECA-VSP could replace SEPARAX-SP without problems.
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.
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.
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.
The stable supply of the cellulose acetate membrane (SEPARAX-SP) for electrophoresis support media became difficult with production device deterioration. Therefore, Toyo Roshi Kaisha, Ltd. was transferred production technology from Fuji Co. and tried to develop a new membrane with similar performance. At the beginning physical properties of the membrane differed slightly possibly due to the difference of production device; however, recently a new membrane with similar performance to SEPARAX-SP has been completed and named SELECA-VSP.Physical properties, such as tearing strength and elasticity, electrophoretic properties of SELECA-VSP were similar to those of SEPARAX-SP. By scanning electron microscope, SELECA-VSP membrane surface and cross section showed similar image and uniformly pored.To investigate a practical use of SELECA-VSP in clinical laboratory, a working group was organized by Japanese Electrophoresis Society and has examined basic and clinical performance of SELECA-VSP. Because the results have been revealed almost good performance, we go on developing for placing SELECA-VSP on the market.
Recently a new cellulose acetate membrane for electrophoresis to analyze protein fractions, named SELECA-VSP, was developed by Toyo Roshi Kaisha Ltd. In advance of commercial supply, a study group was organized to investigate utility of this new membrane in comparison with SEPARAX-SP, a standard membrane for protein fraction assay widely used in Japan. As a member of this study group, our laboratory mainly investigated the difference between the two membranes when analyzing protein fractions in serum as well as ascites, pleural fluid, cerebrospinal fluid and urine. JOKO-CTE8000 was used as a protein fraction analyzer. When buffer lacking EDTA was used in electrophoresis of serum and ascites, the correlation efficiency between the two membranes in percentage of the β fraction was inferior to that of other fractions. When EDTA-containing buffer was used in analysis of serum and ascites, however, the correlation between the two membranes in β-fraction was improved to a comparable level to other fractions. The correlations between the two membranes were satisfactory in analysis of samples other than serum and ascites, regardless of the presence of EDTA in buffer. We conclude that this newly developed membrane has the similar performance to a standard membrane and could be applied in assay of protein fraction in clinical samples.
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.
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.
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.
Glycosaminoglycans were isolated from the equine gastric mucosa. Dowex 1×2 column chromatography was used.
Fractionation of nondialyzable material by Dowex 1×2 column chromatography yielded six fractions at the molarity of NaCl ranging from 0.5 to 2.0mole; they were identified as hyaluronic acid, heparan sulfate, chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively.
A study has been made of glycosaminoglycans in the several regions of the human heart including the right ventricular wall, pulmonary valve, interventricular wall, left ventricular wall and aortic valve, and also pulmonary artery and aorta. The uronic acid content in the aortic valve was approximately 8 times more than that in the ventricular walls.
Cellulose acetate strip-electrophoresis of concentrates from the ventricular walls, pulmonary artery and aorta in barbital buffer revealed the presence of three alcian blue positive fractions, numbered in order of increasing electrophoretic mobility, fraction I, fraction II and fraction III, respectively; the fraction I corresponded in mobility to hyaluronic acid, fraction II to heparan sulfate, and fraction III to chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, whereas in both the aortic valve and pulmonary valve, the fraction II which corresponded with the mobility of heparan sulfate was scarecely found.