Biomedical subjects
M Unoki
Publications and source records attributed to M Unoki.
Enzyme immunosensors based on electropolymerized polytyramine modified electrodes.
Highly sensitive amperometric enzyme immunosensors for human immunoglobulin G (IgG) were prepared on the basis of electrogenerated polytyramine (PTy, tyramine = p-(2-aminoethyl)-phenol) modified electrodes. Properties of PTy films changed depending on electrolysis conditions. On the basis of the found properties of the films, an effective IgG sensor was prepared: a PTy film was formed first from an acid solution on a Pt electrode, and the surface was further covered with a PTy film from an alkaline methanol solution to give a PTy doubly coated electrode on which anti-IgG was then immobilized. This electrode provided a large surface area with little non-specific adsorption of proteins. By means of the competitive enzyme immunoassay technique using glucose oxidase (GOD) labeled IgG conjugates, IgG was determined in the concentration range of c. 10 pg/ml-1 mg/ml from the oxidation current of H2O2 generated by the enzyme (GOD) reaction using the above IgG sensor. Also, an anti-IgG immobilized electrode, prepared by using a Pt electrode singly covered with a PTy film from an alkaline methanol solution, acted as an effective IgG sensor with a detection limit for IgG of c. 100 pg/ml.
Epitope mapping of human thyrotropin.
Epitope mapping of hTSH was carried out using 19 monoclonal antibodies prepared with hTSH or its beta-subunit as antigen. The affinity constants of the monoclonal antibodies ranged from 9.6 X 10(7) to 5.7 X 10(9) mol/l for hTSH. The binding activities of monoclonal antibodies were maintained or in some cases rather enhanced after removal of the sugar moiety of the subunits of hTSH, and completely diminished after reduction of intramolecular S-S bonds in the subunits of hTSH. Ten monoclonal antibodies recognized the epitopes on hTSH (alpha:beta subunit combined form) and on free alpha-subunit form. Eight other antibodies recognized the epitopes on free/or combined form of beta-subunit, all of which did not recognize any other human glycoprotein hormones. The monoclonal antibodies directed against the alpha-subunit could bind also other human glycoprotein hormones to a varying extent. On the basis of results from competitive binding studies, the antibodies directed against alpha-subunit and those against beta-subunit were each classified into five subgroups recognizing different antigenic determinants. The remaining one antibody recognized an epitope expressed only by hTSH and not by the free subunits. In addition, a positive cooperativity on the binding of hTSH was observed between monoclonal antibodies directed towards a particular epitope on the alpha-subunit and those towards a epitope on the beta-subunit. From these data, two-dimensional map of epitopes on hTSH was constructed. The epitopes on each subunit were found to form a cluster with complicated overlapping, suggesting a highly conformational structure.
Highly specific enzyme immunoassay for the beta-subunit of human thyrotropin with use of monoclonal antibodies.
A highly specific enzyme-linked "sandwich" immunoassay is described for determining free human thyrotropin (hTSH) beta-subunit in serum by using a anti-hTSH beta-subunit monoclonal antibody conjugated with beta-D-galactosidase (EC 3.2.1.23) and a solid phase consisting of silicone rods coated with another monoclonal antibody. We could detect as little as 0.04 ng of beta-subunit per assay. The measurable range of hTSH beta-subunit concentrations in serum was 0.4 to 50 micrograms/L. The assay demonstrated little or no cross reactivity with intact hTSH, hTSH alpha-subunit, or human choriogonadotropin. The mean CVs were 12.2% within assay, 13.9% between assay. The hTSH beta-subunit was not detectable in sera from healthy subjects, patients with hyperthyroidism, or two patients with pituitary tumors producing TSH. It was measurable (at concentrations of 0.65 to 2.70 micrograms/L) in sera from eight of 23 hypothyroid patients. In five of the hypothyroid patients examined, the concentration of hTSH beta-subunit in serum increased after administration of thyroliberin. This method may be useful in elucidating the physiological and pathological significance of the hTSH beta-subunit and in examining the function of the hypothalamus-pituitary-thyroid axis.
Plasma phosphorus, alkaline phosphatase and calcium levels, and bone mass of the second metacarpal in women: relation to body weight.
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