[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].
OBJECTIVE
To screen monoclonal antibodies to amylin from a constructed  human phage antibody library and identify their antigenic specificity and combining activities.


METHODS
The heavy chain Fd fragment and light chain of human immunoglobulin   genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR,  and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I,  Xba I,  Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis.


RESULTS
A Fab phage antibody library with 0.8×10(8); members was constructed with    the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen,  specific anti-amylin Fab antibodies were enriched by screening the library   for  three times.  Phage-ELISA assay showed the positive clones had very good   specificity to amylin antigen.


CONCLUSIONS
The successful construction  of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.
Department of Endocrinology, Tianjin Medical University, Tianjin, China. gdedelweiss@163.com
Qian
Q
Gong
Chang-ying
CY
Li
Ji-wu
JW
Chang
Tie-hong
TH
Zhu
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D058228:Islet Amyloid Polypeptide / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D058228:Islet Amyloid Polypeptide; D019151:Peptide Library; C100277:immunoglobulins, Fd