Preparation and characterization of labelled interferon-gamma and the development of radioreceptor assay for interferon-gamma.
A 125I-labelled recombinant interferon-gamma (IFN-gamma) was prepared by the lactoperoxidase-glucose oxidase method. The specific activity of the labelled IFN-gamma was 31 Bq U-1 and its molecular weight, immunoreactivity and receptor binding ability remained the same after the labelling. Using the labelled IFN-gamma and FL5(-1) cells from human amniotic membrane, a radioreceptor assay was developed. Natural IFN-gamma, recombinant IFN-gamma and the labelled IFN-gamma were observed to bind to the same binding sites on the cells with similar affinity (Kd = 1.3-2.2 x 10(-10) M). The radioreceptor assay was more specific than a bioassay because the labelled IFN-gamma did not compete with IFN-alpha or IFN-beta. It was much more sensitive (90 pM) than conventional competitive radioimmunoassay (300 pM) using the same labelled IFN-gamma, and as sensitive as immunoenzymometric assay (60 pM). The radioreceptor assay should be useful not only for research on IFN-gamma but also for the determination of biological activity in process control and/or quality control of IFN-gamma manufacturing.
Kominami
G
G
Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan.
Mori
S
S
Fujimoto
M
M
Kono
M
M
D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis* / Q000378:metabolism; D011863:Radioimmunoassay; D011869:Radioligand Assay / Q000379:methods
D007371:Interferon-gamma