Isolation and characterization of rabbit single chain antibodies to human Reg Ialpha protein.
To investigate the role of Reg Ialpha in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Ialpha. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Ialpha. Anti-Reg Ialpha scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein. Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region. We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria. Immunohistochemical studies using one of our anti-Reg Ialpha scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria. This scFv antibody is now being used for studies of the role of Reg Ialpha in human IBD.
Chi
X Sherry
XS
Department of Pathology and Immunology, Division of Laboratory Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8118, St. Louis, MO 63110, USA.
Landt
Yvonne
Y
Crimmins
Dan L
DL
Dieckgraefe
Brian K
BK
Ladenson
Jack H
JH
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D003106:Colon / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007150:Immunohistochemistry; D051847:Lithostathine; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010189:Pancreatic Juice / Q000737:chemistry; D019879:Paneth Cells / Q000737:chemistry; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D002135:Calcium-Binding Proteins; D007135:Immunoglobulin Variable Region; D051847:Lithostathine; D009419:Nerve Tissue Proteins; C497617:REG1A protein, human; D011993:Recombinant Fusion Proteins