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.
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.
Hofer
Thomas
T
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1203, USA.
Tangkeangsirisin
Wisit
W
Kennedy
Michael G
MG
Mage
Rose G
RG
Raiker
Stephen J
SJ
Venkatesh
Karthik
K
Lee
Hakjoo
H
Giger
Roman J
RJ
Rader
Christoph
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D003429:Cross Reactions / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D058851:GPI-Linked Proteins; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000276:immunology*; D000070816:Nogo Receptor 1; D016716:PC12 Cells; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D014611:Vaccination
D000911:Antibodies, Monoclonal; D000939:Epitopes; D058851:GPI-Linked Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009185:Myelin Proteins; D000070816:Nogo Receptor 1; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C486581:Rtn4r protein, rat