Cloning and characterization of antikeratin human antibodies using a semisynthetic phage antibody library.
Antikeratin autoantibodies (AK auto Abs) are very important elements of the human immune system. To improve the outcome of studies on AK auto Abs, it is necessary to generate antikeratin human monoclonal antibodies. The purpose of present study was to isolate antikeratin human monoclonal antibodies by panning a phage antibody library. A semisynthetic phage antibody library with capacity of 4.0x10(8) members was previously constructed. Panning of the library was performed against human epidermal keratin extracted from psoriatic scales. At the last round of the panning, individual colonies were grown in culture for expression of phage antibodies. Their binding activities and specificities to keratin were determined by ELISA, and positive clones were analyzed by DNA fingerprinting. The selected clones were induced with IPTG to express soluble Fab fragments, which were further characterized by ELISA, immunohistochemistry and Western blotting. Finally, DNA sequencing of the variable regions was performed. A human antibody clone which was able to express soluble Fab fragments and recognize Mr 46,000 keratin (K17) was isolated. DNA sequencing demonstrated that the VH and VL of the antibody came from the human VH1 and Vkappa2 families, respectively. We conclude that phage antibody library technology is a powerful way to generate human monoclonal antibodies. The antikeratin antibody we isolated in the present study would be useful in the research on AK auto Abs.
Wang
Gang
G
Department of Dermatology, Xijing Hospital, Fourth Military Medical University, 710032 Xi'an, China. xjwgang@fmmu.edu.cn
Liu
Yu-Feng
YF
Li
Chun-Ying
CY
Lu
Ning
N
Gao
Tian-Wen
TW
Hua
Bing
B
Wang
Yan
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001435:Bacteriophages / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004817:Epidermis / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007633:Keratins / Q000276:immunology*; D008969:Molecular Sequence Data; D011565:Psoriasis / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007633:Keratins