Generation And Identification Of Human Antibodies Against Amyloid β 1-42 From Human Phage Display Antibody Library

Objective: Alzheimer's disease (AD), by far the single most frequent cause of dementia, affects 4% to 10% of the over 65s, becomes increasingly common with advancing age. The aetiology of AD remains unknown, and no treatments reverse or stabilise the disease. Increasing evidence suggests that a key event in the pathogenesis of AD is the altered production, aggregation and deposition of Amyloid β (A (3 ) peptide, a proteolytic fragment of 40-42 residues derived from neuronal amyloid precursor protein (APP). The longer isoform, A (3 1-42 is the more aggregated form and is more highly correlated than A (3 1-40 with disease and neurotoxicity,. There has been considerable interest in using immunization strategies for clearing excessive A β . Studies in animal models of AD have shown that active immunizations or systemic passive immunizations reduced cerebral plaque load and improved behavioral deficits. The objective of our research is to generate human anti-A 8 1-42 antibodies from human semisynthetic phage display antibody library and to identify their specification and activities.Methods: A β 1-42 was coated on Nunc-immunotube by Glutaraldehyde. Panning from semisynthetic phage antibody library against A (3 1-42 was conducted to select specific antibodies. And the eluted phage was collected and enriched for the next round of selection. The panning and propagating steps were repeated 4 times. The antigen binding activitywas determined by ELISA. And the phage-display ScFv fragments were detected by horseradish peroxidase (HRP)-conjugated anti-M13 antibodies. The phagemid of positive clones was extracted and were transformed into E.coli HB2151. And the bacteria wese induced to give soluble expression of ScFv antibody fragments by isopropyl- |3 -D-thiogalactoside (IPTG). During Western blot, soluble ScFv fragments were detected by primary mouse c-myc antibodies and secondary HRP-conjugated sheep anti-mouse antibodies. Diaminobenizidine (DAB) was used as the final substrateResults: After 4 rounds of panning and propagating, 192 individual clones were selected to test by ELISA, and 56 positive clones were obtained. 8 positive clones were used to infect E.coli HB2151. By the induction of IPTG, soluble ScFv fragments were expressed Western blot analysis showed that the soluble ScFv of Nl clone bound specifically with A 1-42, but not with bovine serum albumin (BSA).Conclusion: Human anti-A 1-42 antibodies can be isolated from phage antibody library without application of immunization, which establishes a basis for diagnosis and immunotherapy of Alzheimer's disease.