[Screening of human anti-gamma-seminoprotein light chain guided with the murine Fd fragment].
OBJECTIVE
To screen human anti-gamma-sm (gamma-seminoprotein) light chain (Lc) with guided selection of murine Fd fragment.


METHODS
The human Lc repertorie genes were amplified by RT-PCR from PBMC in patients with prostate cancer, and cloned into the phagemid vector pComb3X with murine Fd gene against gamma-seminoprotein to construct the human-mouse hybrid Fab antibody library. The size of the library, antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified gamma-sm was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected by ELISA with pIII-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST.


RESULTS
A 1.2x10(7) CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds' panning with gamma-sm, 5 positive clones were selected by ELISA and 2 clones with higher affinity were selected. Sequence analysis suggested these two positive clones contained the same light gene with high V(L) homology to human germline gene IGKV4-1*01.


CONCLUSIONS
Human anti-gamma-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.
Zhang
Qing
Q
Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Zhang
Si-he
SH
Yi
Jing
J
Su
Ming-quan
MQ
Bao
Guo-qiang
GQ
Hao
Xiao-ke
XK
D000818:Animals; D000912:Antibodies, Neoplasm; D000918:Antibody Specificity; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000276:immunology*; D008297:Male; D008403:Mass Screening; D051379:Mice; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D017430:Prostate-Specific Antigen / Q000276:immunology*; D011471:Prostatic Neoplasms / Q000276:immunology*
D000912:Antibodies, Neoplasm; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C100277:immunoglobulins, Fd; D017430:Prostate-Specific Antigen