Journal of Biochemistry Advance Access originally published online on January 2, 2008
Journal of Biochemistry 2008 143(4):475-486; doi:10.1093/jb/mvm239
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© 2008 The Japanese Biochemical Society.
Immunoreactivity of Phage Library-derived Human Single-Chain Antibodies to Amyloid Beta Conformers In Vitro
1Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto 1-21-40, Kagoshima, Kagoshima 890-0065; 2Department of Neuropathology and Neuroscinece, Tokyo University, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033; 3The Second Department of Anatomy, Center of Chronic Viral Disease, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima, Kagoshima 890-8544; and 4Chemo-Sero-Therapeutic Research Institute, Kyokushi, Kikuchi, Kumamoto 869-1298, Japan
*To whom correspondence should be addressed. Tel: +81 99 285 8345, Fax: +81 99 258 4706, E-mail: kazu{at}be.kagoshima-u.ac.jp
Received September 29, 2007; Accepted December 11, 2007
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Abstract |
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The pathogenesis of Alzheimer's disease involves conformational changes of Aβ. A series of antibodies recognizing a distinct conformation of Aβ (snapshot antibody) is useful for both understanding the mechanism of molecular conversion and identifying diagnostic and therapeutic reagents. As Aβ with various conformations can be prepared in vitro under varying physicochemical conditions, snapshot antibodies can be isolated by directly binding to target molecules with antibody-displaying phages. We tested the feasibility of this idea. We show a feature of several Aβ-reactive antibodies isolated from our human single-chain Fv antibody-phage library and particularly report the characteristics of an scFv clone, B6, selected from the fibrillar Aβ1–42-coated biopanning. B6 bound to fibrillar Aβ1–42 as well as globulomer Aβ1–42 but not to soluble Aβ1–42 or Aβ1–40. B6 inhibited Aβ1–42 fibril formation with 600 nM IC50 in spite of being the monovalent scFv form. Epitope analysis suggested that the binding site might be located at the β2 sheet of the C-terminus of Aβ1–42. Although it is believed that N-terminus-recognizing antibodies tend to show the capability to inhibit Aβ1–42 fibrillation, B6 is the first human inhibitory antibody recognizing the C-terminus of Aβ1–42.
Key Words: amyloid beta 1–42, conformation, human antibody, single-chain variable fragment, scFv
Abbreviations: Aβ1–42, amyloid beta 1–42; AP, alkaline phosphatase; BN-PAGE, blue native PAGE; CBB, Coomassie brilliant blue; CDR, complementary determining region; DMSO, dimethyl sulphoxide; FR, framework; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HRP, horseradish peroxidase; IC50, 50% inhibitory concentration; mAb, monoclonal antibody; scFv, single-chian variable fragment; ThT, Thioflavin T; Vh, variable domain of immunoglobulin heavy chain; Vl, variable domain of immunoglobulin light chain