Ishikawa Eiji

An enzyme immunoassay for the measurement of thyroglobulin in human serum

An enzyme-linked sandwich immunoassay using silicone rods coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin G (Fab) conjugated with β-d -galactosidase was developed for the measurement of thyroglobulin in human serum. The volume of serum needed for the assay was as little as 2 μl. The sensitivity of the assay was 3.5 ng/ml, which is equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by the following observations: (1) The absence of crossreaction of thyroxine and triiodothyronine, (2) nondetectability of thyroglobulin in the sera of patients who underwent total thyroidectomy, (3) parallelism of the standard curve with dilutions of reference serum. The precision of the assay was proven by the demonstration of the sufficient recovery of human thyroglobulin added to sera (92–99%) and coefficients of variance in within and between assays were 6.2–9.3 and 2.5–5.3%, respectively. Furthermore, a highly significant correlation was observed between thyroglobulin concentrations measured by our enzyme immunoassay and those by radioimmunoassay (r = 0.99, p < 0.001, n = 63). Human thyroglobulin in serum was detectable in 90% of 146 normal subjects, the concentration (mean ±S.D.) being 13.3 ± 10.3 ng/ml.

A Highly Sensitive Bioluminescent Assay of β-D-Galactosidase from Escherichia coli Using 2-Nitrophenyl-β-D-galactopyranoside as a Substrate

Abstract A highly sensitive bioluminescent assay of β-D-galactosidase from Escherichia coli is described. D-Galactose was released from 2-nitrophenyl-β-D-galactopyranoside as a substrate by the catalytic action of β-D-galactosidase, and subsequently NADH was formed using galactose dehydrogenase. NADH was measured by a bioluminescent assay using NAD(P)H:FMN oxidoreductase and luciferase from Photobacterium fischeri. The detection limits of β-D-galactosidase for 100 and 1,000 min assays were 2 × 10−21 mol and 2 × 10−22 mol, respectively. When the volume of the reaction mixture for β-D-galactosidase assay was reduced from 2 μ to 0.5 μ1, the detection limits were reduced to half.

Demonstration and Measurement of Human Interleukin-1α (hIL-1α) and Interleukin-1β (hIL-10) in vitro and in vivo by Sensitive Enzyme Immunoassay

Abstract hIL-1αa and hIL-1β were demonstrated and measured in culture supernatants of peripheral blood monocytes and mononuclear cells by sensitive and specific enzyme immunoassays. Two components for both hIL-1α and hIL-1β were demonstrated by gel filtration of culture supernatants: one with the same molecular weight as recombinant hIL-1α or hIL-1β and the other with a molecular weight of about 30,000. The production of hIL-1α and hIL-1β increased 1.6 to 38-fold in the presence of Escherichia coli (E. coli) lipopolysaccharide. Levels of hIL-1α and hIL-1β became almost plateau within 24 h in culture supernatants of monocytes but became maximal within 12–18 h and decreased afterwards in culture supernatants of mononuclear cells, suggesting that hIL-1α and hIL-1β were produced by monocytes and consumed or degraded by other cells. In addition, hIL-1α and hIL-1β were detected in sera from patients with various disorders but not in sera from healthy subjects.