Development of monoclonal antibodies against human IRF-5 and their use in identifying the binding of IRF-5 to nuclear import proteins karyopherin-alpha1 and -beta1.
OBJECTIVE
IRF-5 is a direct transducer of virus-mediated and TLR-mediated signaling pathways for the expression of cytokines and chemokines which form homodimers or heterodimers with IRF-7. However, direct IRF-5-specific monoclonal antibodies (mAbs) are not available at present. These could be used to further evaluate the functions of IRF-5. In this study, we produced and characterized three mouse mAbs to human IRF-5. The binding of IRF-5 to nuclear import proteins was first identified using a mAb.


METHODS
His-tagged human IRF-5 protein spanning amino acid residues 193-257 was used as an antigen and three mAbs were produced. The mAbs were tested with ELISA, Western blot analysis (WB), immunofluorescent staining (IF), and immunoprecipitation (IP). In addition, the nuclear import protein which carried phosphorylated IRF-5 was identified using one of these mAbs.


RESULTS
MAbs 5IRF8, 5IRF10 and 5IRF24 which reacted with the recombinant His-IRF-5(193-257) protein were produced. All mAbs bound to human IRF-5, but not to IRF-3 or IRF-7. They could be used for WB, IF, and IP studies. The binding of phosphorylated IRF-5 to karyopherin-alpha1 and -beta1 was also identified.


CONCLUSIONS
Human IRF-5-specific mAbs are produced for studying the immunologic roles related to IRF-5. Phosphorylated IRF-5 is transported to the nucleus by binding to nuclear import proteins karyopherin-alpha1 and -beta1.
Yeon
Soo-In
SI
Department of Microbiology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea.
Youn
Ju Ho
JH
Lim
Mi Hwa
MH
Lee
Hye Ja
HJ
Kim
Young Mok
YM
Choi
Ji Eun
JE
Lee
Jae Myun
JM
Shin
Jeon-Soo
JS
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D050835:Interferon Regulatory Factors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D041681:NIH 3T3 Cells; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D028901:alpha Karyopherins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; C431713:IRF5 protein, human; D050835:Interferon Regulatory Factors; C423142:KPNA1 protein, human; C424166:KPNB1 protein, human; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins