Yoshihiro Ashihara

Sensitive enzyme-linked immunosorbent assay for adult T-cell leukemia-derived factor and normal value measurement.

Four different monoclonal antibodies against recombinant adult T‐cell leukemia‐derived factor (ADF), identical to thioredoxin, were established and used for the determination of ADF concentration in serum. Using two of the monoclonal antibodies, we developed a two‐step enzyme‐linked immunosorbent assay (ELISA) for ADF. This ELISA showed a highly specific reactivity on ADF with no cross‐reactivity to several proteins with homologue sequence on the active center. The detection limit of the assay was 2.0 ng/ml (mean ± 2 SD). The intra‐ and interassay coefficients of variation (CV) were 0.81–3.74% (n = 8) and 4.78–6.97% (n = 7), respectively. The normal value of ADF mean concentration from 145 healthy donors was 40.8 ng/ml.

Simple devices and their possible application in clinical laboratory downsizing.

The pressure to contain medical costs prompted the development of sophisticated downsizing options for the clinical laboratory, such as automation, information systems, or miniaturization of analytical methods. Faced with an array of approaches to choose from, medical institutions have to select the method that can best serve their particular purposes and circumstances. However, the concept of downsizing needs to be revised and applied to the entire medical system. One way to reduce total medical expenditure would be to shift to near-patient or POC testing, through the adoption of simple devices for immunoassay based on immunochromatography. Recent advancements in the area of simple devices have suggested their possible application for emergency testing sites. This paper reviews the specific situation of Japan with respect to laboratory downsizing and market environment, and discusses the possible future applications of simple devices to near-patient or POC testing.

Homogeneous enzyme immunoassay for macromolecular antigens using avidin biotin enzyme

A unique homogeneous enzyme immunoassay used for the determination of haptens and macromolecular antigens has been developed. It consists of avidin–antigen conjugate, anti‐ligand antibody, and biotin enzyme (propionyl‐CoA carboxylase). The immunological reaction of the conjugate with antibodies specific to ligand sterically prevents enzyme inactivation by avidin. Goat IgG and theophylline were used as model antigens. Avidin–theophylline conjugate inhibited 100% of enzyme activity. Avidin goat IgG conjugate inhibited 90% of enzyme activity.

Development of a highly sensitive enzyme immunoassay for human calcitonin using solid phase coupled with multiple antibodies

We have developed a highly sensitive chemiluminescent enzyme immunoassay for human calcitonin using three different mouse monoclonal antibodies that recognize the N-terminal, C-terminal and central portions of a human calcitonin molecule. The assay signal in a two-step sandwich enzyme immunoassay for human calcitonin using a solid phase coupled with a mixture of monoclonal antibodies, CT08 and OCT1, was 3·7-fold higher than when using either or both solid phases coupled with CT08 or OCT1, respectively. This enhancement is the result of improved avidity of immobilized antibodies and greater stability of the complex of immobilized antibodies and calcitonin in the first reaction, which resulted in greater reactivity of the immunocomplex with alkaline phosphatase-conjugate in the second reaction. The present assay showed a linear response up to 2·5 μg/L of human calcitonin and a high specificity for human calcitonin, but not for rat calcitonin, human calcitonin gene-related peptide and rat calcitonin gene-related peptide. The detection limit of human calcitonin was estimated to be 0·29 ng/L at zero (assay blank) + 3 SD. Interbatch coefficients of variation ranged from 2·2–26·7%.

STUDIES ON THE INITIAL REACTIVE SITES FOR PLATELET ACTIVATION

The binding sites for cationized ferritin or ferritin‐conjugated wheat germ agglutinin on the cell surface were studied on platelets before or after fixation in glutaraldehyde. The effects of neuraminidase on the binding sites were also demonstrated after fixation of the platelets. Changes in the binding sites and distribution pattern due to exposure to these ligands were further investigated in the unfixed platelets under a variety of conditions such as incubation time and medium. The fixed platelets incubated with either ligand showed an even and continuous distribution of particles on the cell surface. In the unfixed platelets, the ligands were rapidly moved and aggregated probably by lateral migration after binding to the cell surface. The ligands were also bound to the membrane surface and simultaneously appeared in the interior of the open canalicular system. As the binding sites were moved on the cell surface as well as into the open canalicular system, morphological changes suggestive of platelet secretion occurred. The binding sites of either ligand were redistributed on the platelet cell surface. Glycoprotein lb, thought to be the receptor site for wheat germ agglutinin on the cell surface, contains sialic acids that contribute to the negative charge of platelets. Therefore, glycoprotein lb may play an important role as the initial reactive site for thrombotic stimuli.

Micro Multiple Immunoassay Using Silicon-Based Microfabricated Protein Biochip

We have developed multiple parallel immunoassay using silicon-based protein chip for parallel quantification of specific proteins. We employed micro fabricated chips with 10,000 pillars per square centimeter. The chip is composed of six lanes with 5x50 spotting sites. Several target proteins, i.e., alpha-fetoprotein (AFP), IL-6, MAPK and H-Ras were measured by fluorescent sandwich immunoassay. Positive signals were obtained simultaneously in each reaction site on the chip. Furthermore, IL-6 showed dose-dependent signals ranging with 5pg/mL to 50ng/mL.