| Research Abstract |
In the present study, IgG was highly purified from pooled sera of healthy volunteers. Galactose-free IgG was then prepared from the human IgG by enzymatic oligosaccharide-remodelling. Analyzes of the protein moiety and oligosaccharide structures of both intact IgG and the oligosaccharide-remodelled IgG indicated that the oligosaccharide-remodelling completely removed galactose residues of IgG without damaging the protein moiety. Using this galactose-free IgG,novel serum assay system for detection of rheumatoid factor was designed as follows. The galactose-free IgG was immobilized on a 96-well microtiter plate, to which serum sample was added. If rheumatoid factor exists in the serum sample, it binds to galactose-free IgG on the plate. Them galactose residues of rheumatoid factors bound to the galactose-free IgG were detected with HRP conjugated lectin. Conditions for immobilizing the galactose-free IgG on the plate, a kind and concentration of the lectin, detergent, and blocking reagent, amount of serum sample analyzed, reaction temperature, and reaction time were investigated in detail. Finally, an optimal assay condition was established for specific detection of oligosaccharides of rheumatoid factor in sera from patients with rheumatoid arthritis. When serum samples from healthy individuals, patients with rheumatoid arthritis, rheumatic diseases, and the other diseases were analyzed using this novel serum test, it was indicated that this novel serum test is superior to conventional serum tests (RA test and RAHA test) for detection of rheumatoid factor in terms of specific detection of patients with rheumatoid arthritis including seronegative patients and early-phase patients and understanding of the disease activity in patients. Therefore, this novel serum test could be very useful for accurate diagnosis and treatment of this disease.
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