Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer.
OBJECTIVE
To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.


METHODS
Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected.


RESULTS
The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6). The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer.


CONCLUSIONS
The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.
Wu
B P
BP
Institute for Digestive Medicine, Nanfang Hospital, Guangzhou 510515, Guangdong Province, China. bpwu@263.net
Xiao
B
B
Wan
T M
TM
Zhang
Y L
YL
Zhang
Z S
ZS
Zhou
D Y
DY
Lai
Z S
ZS
Gao
C F
CF
D000906:Antibodies / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000276:immunology*; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D019151:Peptide Library
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library