[Construction and characterization of anti-DOTA-Y phage antibody library].
OBJECTIVE
To construct a anti-dodecane-tertraacetic acid-yttrium(DOTA-Y) immune Fab phage antibody library.


METHODS
BALB/c were immunized with BSA-Y-DOTA which was prepared by DOTA-conjugated BSA and chelated with Y. After determination of anti-serum, total RNA was extracted from splenic lymphocytes of immuned mice. The heavy chain Fd and light chain Kappa genes repertoires of immunoglobulin were amplified respectively by RT-PCR, and then the amplified products were cloned into the reconstructive phage vector pComb3M to construct anti-DOTA-Y Fab antibody. And then, the recombination rate, diversity and display of Fab antibody library were identified by restriction endonuclease digestion, DNA sequencing and ELISA.


RESULTS
BSA-Y-DOTA was prepared successfully, and a higher titer of immune sera was achieved. The amplified gene fragments of Fd and Kappa chain by RT-PCR were correct and the length was with about 650 bp, and were inserted exactly. The sink size of Fab phage display library reached 8 x 10(7), the re-combination rate was about 90%, and it possesed great diversity. In addition, ELISA detection showed that there was Fab expression on the phage library.


CONCLUSIONS
An immune Fab phage antibody library of DOTA-Y has been constructed successfully, which lays a solid foundation for screening specific anti-DOTA-Y antibody.
Song
Fei
F
Cell Engineeing Research Center, Fourth Military Medical University, Xi'an 710032, China. chcerc1@fmmu.edu.cn
Xing
Jin-liang
JL
Zhang
Si-he
SH
Yang
Xiang-min
XM
Chen
Zhi-nan
ZN
D000818:Animals; D000906:Antibodies / Q000235:genetics*; D006571:Heterocyclic Compounds / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009942:Organometallic Compounds / Q000276:immunology*; D019151:Peptide Library; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012710:Serum Albumin, Bovine / Q000276:immunology
D000906:Antibodies; D006571:Heterocyclic Compounds; D007140:Immunoglobulin Fab Fragments; D007145:Immunoglobulin kappa-Chains; D009942:Organometallic Compounds; D019151:Peptide Library; C113625:yttrium(III)-1,4,7,10-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid; D012710:Serum Albumin, Bovine