Tohoku J. Exp. Med., 2010, 220(3)

Evaluating the Role of Rheumatoid Factors for the Development of Rheumatoid Arthritis in a Mouse Model with a Newly Established ELISA System

YUKI TANAKA,^1,2 HIROAKI KOMORI,¹ SHIRO MORI,³ YOSHIKO SOGA,¹ TAKAHITO TSUBAKI,¹ MIHO TERADA,¹ TATSUHIKO MIYAZAKI,¹ TAKAHIRO FUJINO,² SATOSHI NAKAMURA,⁴ HIROYUKI KANNO,⁵ TATSUYA SAWASAKI,⁶ YAETA ENDO⁶ and MASATO NOSE^1,7

¹Department of Pathogenomics, Ehime University Graduate School of Medicine, Ehime, Japan
²Integrated Center for Sciences, Ehime University, Ehime, Japan
³Department of Oral and Maxillofacial Surgery, Tohoku University Graduate School of Dentistry, Sendai, Japan
⁴Department of Bioengineering, Tokyo Institute of Technology, Tokyo, Japan
⁵Department of Pathology, Iwate University School of Medicine, Morioka, Japan
⁶Cell-Free Science and Technology Research Center, Ehime University, Ehime, Japan
⁷Proteo-Medicine Research Center, Ehime University, Ehime, Japan

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.

keywords —— MRL/lpr; recombinant inbred strain; MXH/lpr; GST; IgG-Fc

© 2010 Tohoku University Medical Press

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Tohoku J. Exp. Med., 2010, 220, 199-206

Correspondence: Masato Nose, M.D., Ph.D., Department of Pathogenomics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.