TY - JOUR
T1 - Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector
AU - Hofer, Thomas
AU - Tangkeangsirisin, Wisit
AU - Kennedy, Michael G.
AU - Mage, Rose G.
AU - Raiker, Stephen J.
AU - Venkatesh, Karthik
AU - Lee, Hakjoo
AU - Giger, Roman J.
AU - Rader, Christoph
N1 - Funding Information:
This research was supported by (i) the Intramural Research Program of the Center for Cancer Research, NCI, NIH (C. R.), (ii) the Intramural Research Program of NIAID, NIH (R. G. M.), (iii) NINDS, NIH grant NS047333 (R. J. G.), and (iv) the New York State Spinal Cord Injury Research Program (R. J. G. and C. R.). T. H. was the recipient of a postdoctoral fellowship from the Schweizerische Stiftung für medizinisch-biologische Stipendien. W. T. was a visiting scientist funded by the Royal Thai Government. K. V. was supported by NIH predoctoral fellowship NRSA F31 NS049870. We thank Dr. Barbara A. Newman and Cornelius B. Alexander for rabbit immunization and tissue collection, Reema R. Sikka for assisting in library generation and selection, Dr. Sivasubramanian Baskar for supporting flow cytometry studies, Lisa Boyd and Dr. David H. Margulies for supporting BIAcore studies, and Dr. Stephen J. Stahl for reading the manuscript.
PY - 2007/1/10
Y1 - 2007/1/10
N2 - NgR1, NgR2, and NgR3 which constitute the Nogo-66 receptor family are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we previously reported the generation of selective rabbit polyclonal antibodies. Here we exploit the same immune repertoires by phage display technology to generate rabbit monoclonal antibodies (mAbs) with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Using immunofluorescence microscopy and immunoprecipitation, we demonstrate strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG. The rabbit mAbs reported here together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply which will aid investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS.
AB - NgR1, NgR2, and NgR3 which constitute the Nogo-66 receptor family are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we previously reported the generation of selective rabbit polyclonal antibodies. Here we exploit the same immune repertoires by phage display technology to generate rabbit monoclonal antibodies (mAbs) with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Using immunofluorescence microscopy and immunoprecipitation, we demonstrate strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG. The rabbit mAbs reported here together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply which will aid investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS.
KW - Nogo-66 receptor family
KW - Phage display
KW - RTN4R
KW - RTN4RL2
KW - Rabbit monoclonal antibodies
KW - b9 allotype
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U2 - 10.1016/j.jim.2006.10.007
DO - 10.1016/j.jim.2006.10.007
M3 - Article
C2 - 17140598
AN - SCOPUS:33845881958
SN - 0022-1759
VL - 318
SP - 75
EP - 87
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -