Kazuo Hosoi

An enzyme-linked immunosorbent assay for the measurement of human interleukin-6

An enzyme-linked immunosorbent assay has been developed to measure human interleukin-6. The assay, based on the avidin-biotin amplified two-step sandwich method, is quick (requiring 4.5 h), sensitive (detecting 9.5 pg/ml) and satisfactory in reproducibility and specificity. It shows good correspondence with the results of bioassays, and it is not affected by serum and plasma components. These results indicate that this ELISA is suitable for application to clinical samples, which is a major advantage over the widely used bioassays.

Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lys171 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.

Characterization of four different mammalian-cell-derived recombinant human interferon-β1s

In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.