Enrichment From The Phage Scfv Human Antibody Library Screening Of Anti-a¦Â Antibody Gene And Preliminary Identification

Alzheimer's Diseases (AD) , characterized with amyloid fiber or senile plaques and neurofibrillary tangles in pathological changes, is a kind of neural degenerative disease that make people disremember about recent things and often occur in ages. Since the end of last century, China has become an ageing society in advance. Alzheimer's Disease is becoming the fourth mortality cause of human being secondary to heart disease, tumor and brain stroke. Up to now, the diagnosis and therapy of Alzheimer's Disease remain a problem. The studies on mechanism of this disease display that Aβ(Amyloid-Ppeptide)and it's aggregation may be main pathogenic substance. Immunological treatment is a new therapeutic approach for treatment of AD.The library was screened by Aβ to get the antigen-specific phage clones .Above all, four panning rounds were performed of the library with Ap. The transformed cells were infected with M13K07 helper phage to produce a phage form of library. Then the library was added into immunotubes coating with Aβ and incubated for a period of time and washed several times. The phage clones binding specifically to Aβ were retained in the tubes. These clones were eluted from the wells using Gly-HCl (pH 2.2) buffer and infected log phase E.coli TGI to amplify antigen-specific phage clones. Repeat the above four times. After four panning rounds of "binding-elution-amplification", the clones binding specifically to Ap in library increased from 5.1 ×10^-5 pfu after the first round of panning to 1.38×10^-2 pfu at the end. A large enrichment of 271 fold was observed.75 bacterial clones were randomly picked up and infected with M13KO7 helper phage to display scFv on the surface of phage. The positive anti- Aβ phage clone was screened with ELISA. The results showed that 5 positive clones had remarkable binding affinity with the highest OD₄₉₀...