Cloning and characterization of antikeratin human antibodies using a semisynthetic phage antibody library

Abstract

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.

Keratin 17: A Critical Player in the Pathogenesis of Psoriasis

“…In 2004, our group cloned and expressed anti‐K17 human monoclonal antibodies by panning a semisynthetic phage antibody library against human epidermal keratins extracted from psoriatic scales . In the following year, we discovered three new immunodominant T‐cell activating epitopes on K17 in addition to the “ALEEANxxL” amino acid sequence (Fig.…”

Reply

“…These two kinds of antibodies, especially the latter one (the genetically engineered antibody), could penetrate the living keratinocytes and showed strong potential to inhibit the proliferation of cultured keratinocytes in a dose-dependent manner. [7][8][9][10] Specifically, small interfering RNA targeting K17 inhibited the proliferation of a human keratinocyte cell line keratinocytes and sensitized these cells to apoptosis based on our data. 11 The results mentioned above made us assume that K17 may be a new target that could be used for psoriasis treatment, as hyperproliferation of keratinocytes is the most obvious feature in psoriatic epidermis.…”

“…Kelp is recognized as causing an acneiform eruption 8 where the iodide source is indeed high, and there is little doubt that a papular and papulopustular acneiform eruption can be triggered by halides, but the hallmark of acne, the comedo, is not part of the initial lesion in this picture. Comedones may appear as secondary lesions.…”

Blockade of Autoantibody-Initiated Tissue Damage by Using Recombinant Fab Antibody Fragments against Pathogenic Autoantigen

“…The phagemid libraries were electroporated into E. coli XL1-Blue strain (Stratagene, La Jolla, CA), and the phage display of the libraries was performed as described elsewhere. 17,20 Before amplification, the resulting libraries were examined for the coexpression of heavy and light chains by enzyme digestion and for the diversity by fingerprinting of antibody genes (Fd and light chain) of 24 randomly selected single colonies. 20,21 The amplified recombinant phages were purified from culture supernatants by polyethylene glycol precipitation and resuspended in PBS, pH 7.4, containing 1% bovine serum albumin (BSA) and 10% glycerol.…”

“…After neutralization with 1 mol/L Tris, pH 7.4, eluted phages were used to infect a fresh culture of XL1-Blue E. coli, which was amplified overnight as previously described. 20 Phages were harvested from culture supernatants and then repanned against rhNC16A for three subsequent rounds as described for the original library. Individual single ampicillinresistant colonies resulting from infection of E. coli XL1-Blue with the eluted phage from the fourth panning round were isolated, and the binding to rhNC16A was confirmed by ELISA using HRP-conjugated anti-M13 mAb (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK) as the developing reagent.…”