Ken Sugo

Development of a particle agglutination assay system for detecting Japanese encephalitis virus-specific human IgM, using hydroxyapatite-coated nylon beads.

Japanese encephalitis virus-specific IgM is a reliable indicator for serodiagnosis of Japanese encephalitis. A particle agglutination (PA) assay system was developed to detect anti-Japanese encephalitis virus IgM in human serum samples. The newly developed PA assay consisted of hydroxyapatite-coated nylon beads and V-bottom 96-well microplates. Hydroxyapatite-coated nylon beads were coated with Japanese encephalitis virus antigens. Japanese encephalitis virus antigen-coated, hydroxyapatite-coated nylon beads agglutinated in the IgM-captured wells when anti-Japanese encephalitis virus IgM-positive serum samples were used. A button pattern was formed at the bottom of the wells when anti-Japanese encephalitis virus IgM-negative serum samples were used. Thirty anti-Japanese encephalitis virus IgM-positive serum samples from Japanese encephalitis-confirmed cases were tested by the PA assay. All these serum samples were determined to be Japanese encephalitis virus IgM-positive. IgM titers determined by the PA assay corresponded to those determined by enzyme-linked immunosorbent assay. The titers were consistent in two independent PA assays. These results indicate that the newly developed PA assay is a reliable method for detecting anti-Japanese encephalitis virus IgM in human serum samples and that this assay will be a suitable diagnostic system especially in rural areas of Asia.

Preparation of Polyethylenimine-Hydroxyapatite and its Chromatographic Use

Abstract Hydroxyapatite (HAp-CHT TypeII 40 µm) was coated with a stable layer of polyethylenimine (PEI). The resulting material was useful in column chromatography of acidic materials. A mixture of AMP, ADP, ATP, and adenosine could be resolved readily. The convenient separation of ovalbumin (one of the phosphoproteins) from other standard protein mixtures was observed.

Hydroxyapatite chromatographic procedures for phospholipids

ABSTRACT Monophosphates such as the phospholipid lecithin poorly adsorb to hydroxyapatite because of anionic repulsion with hydroxyapatite phosphate groups. Here, we attempted to optimize the adsorption and elution of lecithin from hydroxyapatite chromatographic columns. The addition of Ca2+ ions to the running buffer masked the hydroxyapatite phosphate groups and increased the adsorption of lecithin from 38.9% to 96.1%. The stably adsorbed lecithin was eluted with isopropanol at 99.1% efficiency. By establishing an isopropanol gradient using 2-morpholinoethanesulfonic acid buffer supplemented with Ca2+ ions, the chromatographic method developed here may be suitable for the purification of various phospholipids, including monophosphates.

Hydroxyapatite chromatographic procedures for phospholipids: Endotoxin purification methods

ABSTRACT We studied the one-step purification of endotoxin by hydroxyapatite chromatography using total lipids extracted from Escherichia coli by the Bligh and Dyer method. High endotoxic activity was detected in a fraction eluted from 0.5 mg total lipids using an isopropanol gradient under salt-free buffer conditions. The fraction contained at least 7,000 endotoxin units and was completely free from other phospholipids. Notably, the running time of the chromatographic separation was approximately 4 h, whereas purification by the conventional hot phenol–water procedure requires ≥60 h. The procedure developed here is a simple, scalable, and rapid method for the purification of endotoxin.