Engineering the male-specificity of Fab against SDM antigen by chain shuffling

High-titer serologically detected male (SDM) antibody fragments are essential for specific binding to the SDM antigen and promoting its application. The A8 clone previously obtained from an original phage antibody library was further affinity-matured by light- and high-chain shuffling respectively,...

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Bibliographic Details
Published in:Theriogenology 2013-05, Vol.79 (8), p.1162-1170
Main Authors: Wang, Naidong, Yuan, Anwen, Deng, Zhibang, Yang, Qing, Ma, Jun, Tan, Qinghui, Zhang, Sizhe, Xue, Liqun, Cui, Shuliang
Format: Article
Language:English
Subjects:
amino acid sequences
Aminoacridines - immunology
Animals
Antibody repertoire
Antibody Specificity - genetics
antigens
Antigens - immunology
bacteriophages
binding capacity
Cells, Cultured
Chain shuffling
clones
Cloning, Molecular - methods
enzyme-linked immunosorbent assay
Female
Gene Rearrangement - genetics
Immunoglobulin Fab Fragments - biosynthesis
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Fab Fragments - metabolism
Male
Mice
Mice, Inbred C57BL
mutation
Phage display library
Protein Engineering - methods
Recombinant antibody
Recombination, Genetic - physiology
SDM antigen
Sex Characteristics
splenocytes
testes
Western blotting
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Summary:High-titer serologically detected male (SDM) antibody fragments are essential for specific binding to the SDM antigen and promoting its application. The A8 clone previously obtained from an original phage antibody library was further affinity-matured by light- and high-chain shuffling respectively, to generate the end product B9 clone. The binding capacity of B9 phage Fabs to male splenocytes doubled the value of its parental A8 clone (determined using ELISA). Based on immunofluorescent staining, B9-Fabs mainly bound to the surface antigen of male splenocytes and recognized testicular cells. The resulting B9-Fabs detected a single protein (approximately 40 kDa determined using Western blot analysis of male splenocytes and testis); its high SDM antigen binding ability might have been because of mutation sites and varied lengths of the amino acid sequences in the complementarity determining regions-3 of the κ and Fd chains. The new recombinant clones of Fab that were phage-enhanced using chain shuffling were candidate molecules for investigating molecular mechanisms of SDM antigens specific binding and applications.
ISSN:0093-691X
1879-3231
DOI:10.1016/j.theriogenology.2013.02.012