Evaluating the role of rheumatoid factors for the development of rheumatoid arthritis in a mouse model with a newly established ELISA system.
Enzyme-linked immunosorbent assays (ELISA) have been widely used to determine quantitatively autoantibodies. However, the processes for the purification and immobilization of antigens in conventional ELISA methods include multiple steps, which have hampered the application for screening of autoantibodies. Here, we have developed a novel ELISA system using the plates pre-coated with glutathione casein to capture recombinant proteins fused to N-terminal glutathione S-transferase (GST). The GST-fused proteins were synthesized with the wheat germ cell-free protein production system. Thus, the present system combined the GST-capture ELISA with the cell-free protein production system, which allowed immobilization of the recombinant proteins with one-step purification. Using this ELISA method, we determined whether rheumatoid factors (RF), which have been considered as one of the representative disease-specific autoantibodies for rheumatoid arthritis (RA), were genetically associated with severity of arthritis in a mouse model for RA, MRL/Mp-lpr/lpr (MRL/lpr). GST-fused human IgG1-Fc (GST-Fc), synthesized with the robotic protein synthesizer, were used as reactants for RF. Serum samples for RF were prepared from 11 lines of a recombinant inbred mouse strain, MXH/lpr, which was established from intercrosses between MRL/lpr and non-arthritic C3H/HeJ-lpr/lpr (C3H/lpr) strains, composed of a different genomic recombination derived from the parental strains in each line. A correlation of RF titers with the severity of the arthritis in these lines was not significant, indicating genetic dissociation of RF from arthritis and that RF is not necessarily required for the development of RA. The present method may provide high-throughput screening for determining the disease-specific autoantibodies in autoimmune diseases.
Tanaka
Yuki
Y
Department of Pathogenomics, Ehime University Graduate School of Medicine, Ehime, Japan.
Komori
Hiroaki
H
Mori
Shiro
S
Soga
Yoshiko
Y
Tsubaki
Takahito
T
Terada
Miho
M
Miyazaki
Tatsuhiko
T
Fujino
Takahiro
T
Nakamura
Satoshi
S
Kanno
Hiroyuki
H
Sawasaki
Tatsuya
T
Endo
Yaeta
Y
Nose
Masato
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000276:immunology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012217:Rheumatoid Factor / Q000097:blood* / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012217:Rheumatoid Factor