Eiichi Tokunaga

High-performance ion-exchange chromatography of plasma proteins

The use of anion-exchange high-performance liquid chromatography columns for the analysis of plasma proteins has been investigated. Mono Q and Polyanion SI were used as anion-exchangers. Several factors, including solvent composition, pH, flow-rate, sodium chloride linear concentration gradient and sample loading capacity, were examined for their effects on the resolution of protein standards and pooled human plasma (PHP). PHP was separated into ten or more protein fractions by a Mono Q column (50 X 5 mm I.D., flow-rate 2 ml/min) within 10 min. Components analysis of each fraction was performed using immunochemical methods and size-exclusion high-performance liquid chromatography. The Mono Q column was applied to the analysis of IgG myeloma and other patient plasma samples.

Physical and Chemical Changes of ACD-Preserved Blood: A Comparison of Blood in Glass Bottles and Plastic Bags

Abstract. ACD blood was preserved in glass bottles with or without aeration and in plastic bags in air or nitrogen gas at 4.5°C. The blood was examined for physical and chemical changes of erythrocyte membrane resistance, hemoglobin in the plasma, the viscosity of the blood, pH of plasma, and ATP and 2,3‐DPG content of erythrocytes. The blood preserved in plastic bags showed less changes than blood in glass bottles. The presence of air or nitrogen gas in blood seems to increase the pH perhaps by elimination of carbon dioxide (CO2) which in turn causes the different rates of glycolysis in the erythrocytes.

Analysis of Peptic Fragmentation of Human Immunoglobulin G Using High-Performance Liquid Chromatography

Abstract High-performance liquid chromatography using a hydrophilic hard-gel TSK-G3000SW column was employed to investigate the peptic fragmentation of a commercial human immunoglobulin (IgG) and its components, i.e., monomeric, dimeric, and aggregated IgG. The monomeric IgG was digested to yield two major fragments, F(ab′)2 and pFc′, and at least six minor fragments. The dimeric IgG was digested to give three major fragments including F(ab′)2, pFc′, and a fragment similar to F(ab′)2 dimer. The main product from the aggregated IgG was an unknown fragment, and the F(ab′)2 fragment was scarcely observed. These observations demonstrated that three IgG components were different from each other in the susceptibilities to peptic digestion and also suggested that the dimer and aggregates were associated mainly through their Fab regions. The TSK-G3000SW column was very useful as a means of fractionating monomer, dimer, and aggregates of IgG as well as a potential means of analyzing the peptic fragment of IgG.

A Monoclonal Antibody with Broad Reactivity to Human Interferon-α Subtypes Useful for Purification of Leukocyte-Derived Interferon

A monoclonal antibody to human interferon‐α, termed HT‐1 antibody, with a broad reactivity to various subtypes of interferon‐α was prepared. It bound and neutralized all of the four subtypes of E. coli‐derived human recombinant interferon‐α (aL1, aL2, aL4, and aL6) tested; it also neutralized human natural leukocyte interferon but only partially. Human interferon‐β and ‐γ were not bound. The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus. The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2 ′ 108 international units/mg protein). The purified interferon showed, in SDS‐polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol. wt. range of 17,000–22,000, which completely agreed with the profile shown by polyclonal antibody‐purified interferon. Such purified leukocyte interferon‐α preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.

Two-dimensional separation system for analysis of proteins employing isoelectric focusing and high-performance liquid chromatography

Abstract A new type of separation system, which combined isoelectric focusing with high-performance liquid chromatography, was designed for the analysis and fractionation of serum proteins. For the first dimensional separation, carrier-free isoelectric focusing was used to separate proteins according to their electric charge. For the isoelectric focusing, an instrument which consisted of multiple chambers was deviced. For the second dimensional separation, high-performance gel permeation chromatography was used to separate proteins according to their molecular size. Human serum was subjected to analysis with this two-dimensional system, and separation of serum proteins according to their p I and molecular size was demonstrated.

Osmotic Fragility Changes in Preserved Blood: Measurements by Coil Planet Centrifuge and Parpart Methods

Abstract. The coil planet centrifuge (CPC) can be used to measure the osmotic fragility of erythrocytes. Fragility measured by this method alters when different salts are used. The CPC and Parpart methods were used to measure the changes during storage in red cell osmotic fragility in ACD or CPD blood with or without adenine. More marked changes were detected by the CPC method, especially in old cells. The changes of fragility of erythrocytes during storage seem to occur mainly in old cells. Adenine is effective in preventing such changes.

Change of Oxygen Affinity of Hemoglobin in Different Conditions of Blood Preservation

Abstract. Changes of oxygen affinity of hemoglobin were studied in blood preserved in different conditions. Changes in hemoglobin function were least in CPD blood in plastic bags, and more extensive in ACD blood in plastic bags, ACD packed red blood cells in plastic bags and ACD blood in glass bottles, respectively. These decreases are consistent with the changes of organic phosphates levels during storage found in earlier studies. When old erythrocytes were incubated with inosine, pyruvate and phosphate, defective oxygen transport function was completely restored even after 7 weeks storage in ACD.

Report on the 1990th year's annual meeting of workshop for red cell grouping test within the jurisdiction of Central Blood Center, the Japanese Red Cross Society. Partial D antigen of Rh system.

The 1990th years annual meeting of workshop on red cell grouping test within the jurisdiction of Central Blood Center, The Japanese Red Cross Society, has been opened.From the 4 blood centers, 4 blood preparations, each of which was suspected as “partial D” red cells, were submitted as the test samples.On the D antigen, judgement of operators agreed with each other for 3 out of the 4 preparations. But on remained 1 preparation (sample I), judgements of them were not agreed. On this sample I, agglutination reaction by monoclonal anti-D antibody of JRC was remarkably weak but surely observed. As the results, “Partial D” as the suspected antigenicity, was proved to be false, rather, “weak D” on some specific epitopes was suitable on this sample I, presumably.On the otherhand, one preparation which was suspected to have antigenic variation on E antigen was found.