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The Preparation of Human Antibodies Against Aβ

Author: SunChao
Tutor: YuZuoYuan
School: PLA Military Academy of Medical Sciences
Course: Genetics
Keywords: Alzheimer's disease β - amyloid protein Antibody Epitope
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 65
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Abstract

Alzheimer's disease (Alzheimer's disease, AD) is a multi-degenerative disease of the central nervous system in the older age groups, the main clinical features of memory loss, cognitive and motor function disorders. With the intensification of the global population aging, the incidence of the disease of Alzheimer's disease are rising, which triggered a series of social problems are increasing. For the pathogenesis of AD is still a lack of effective treatment, and therefore has important significance to the development of the effects of drugs to treat AD. Not very clear at this stage about the pathogenesis of AD, wherein the β amyloid protein (β-amyloid protein, Aβ) gathered by the normal physiological conditions of the soluble monomeric form is considered to be for the neurotoxicity of amyloid deposition in the AD pathogenesis key events. Studies have shown that: Aβ common pathway of a variety of factors that cause Alzheimer's disease, is a key factor in the pathogenesis of AD and the development of, Aβ is generally considered to be an effective target molecules for the treatment of AD. Aβ is mainly exists in the brain tissue, β-amyloid-like precursor protein (β-amyloid precursor protein, APP) formed through the β pathway. Composition, generally 39 to 43 amino acids having a β-pleated sheet secondary structure of the whole molecule rendered hydrophobic, easily aggregate formation of insoluble deposits. In 1999, Schenk Aβ1-42 on the first application of AD model mice for active immunization breakthrough progress. Thereafter, reports confirm that Aβ peptide and its antibodies can be applied to AD immunotherapy, and be able to get a very good therapeutic effect. Aβ1-42 vaccine developed by Elan Corporation Betabloc (AN1792) after a phase I clinical trial and found that the Betabloc can effectively stimulate the immune response to produce the Aβ amyloid deposition binding antibody can effectively activate microglia clear Aβ starch formation of plaque deposition, and significantly improve patients' cognitive function. Unfortunately, in Phase II clinical experiment, some patients with brain inflammation occurs, the test was forced to terminate. At the same time has made tremendous progress in the passive immunotherapy for AD, the study confirmed that the antibody can effectively application of anti AβN end to improve related symptoms of AD, and will not trigger the immune response of Th1 cells. Elan and Wyeth jointly developed humanized antibody Bapineuzumab anti-AβN end is the first to develop antibodies for the treatment of AD, the ongoing Phase III clinical trials. In this study, we hope to Aβ1-42 and its N-terminal (Aβ1-12, Aβ1-15) to target molecules by bacterial screening combined with solid-phase screening screening of large human phage antibody library screening to obtain specific antibodies, to lay the foundation for the development of AD therapeutic antibody. First of all, this study, in order for Aβ1-12 antibody screening and specific antibodies for screening preliminary epitope identified epitope peptides according to the characteristics of Aβ1-42 molecule structure and bacterial flagella surface display system Aβ1-42 is divided into the Aβ1-12, Aβ12-23, Aβ21-32, Aβ31-42 four epitope peptides were constructed four peptides with bacterial flagellin fusion expression recombinant plasmid can be expressed, and the use of bacterial flagella surface display system were expressed in E.coli GI826 flagella. SDS-PAGE and Western blot and immunofluorescence identified and successfully displayed on the surface of the E.coli GI826 the four peptides Aβ epitopes flagellin fusion expression. Secondly, we Aβ1-42 with its N-terminal (Aβ1-12, Aβ1-15) a high-throughput screening of human antibody. The AD passive immunization Institute of application antibody for murine antibody, limiting its application in the human body. Antibodies obtained from human antibody library to avoid this problem, be directly applied to the human body without human transformation. The present study we were used for the Aβ1-12 bacteria deducted screening for Aβ1-42 solid-phase screening bacteria for Aβ1-12 solid-phase binding filters, as well as four screening programs for Aβ1-15 solid-phase screening The storage capacity of up to 1.35 × 1010 capacity fully synthetic single-chain antibody phage library specific antibody screening, high-throughput screening of antibody. Randomly selected in the screening process, approximately 2000 clones were identified, including bacterial screening clones positive rate was low, solid-phase screening clone positive rate of up to 50%, but due to the solid phase screening for Aβ1-42 full-length screening to obtain specific antibodies may be combined with an arbitrary region of Aβ1-42. Quality antibody screening order for AβN end, we used the first for the Aβ1-12 the bacterial net of filters and then apply the Aβ1-42 to the solid-phase screening programs for Aβ1-12 bacteria solid phase binding screening and cloning positive rate up to 40%. At the same time, we have solid-phase screening for Aβ1-15, but a lower rate of cloning positive. Ultimately, we in bacterial screening process, a specific phage antibody G10. Aβ1-42 solid-phase screening process, two specific phage antibody 18,26. Bacterial solid combination of screening process, we obtained two specific phage antibody H9, B5. Aβ1-15 solid-phase screening process to obtain a specific antibody 87. 18 and H9 with an antibody, this result proved that our program is reliable and effective. Again, we will get specific single-chain antibody transform the form of whole antibodies and antibody transient expression of specific identification. In order to obtain a better stability of the antibody form, we have obtained for the screening of five specific phage antibody forms of transformation of the whole antibody, and five whole antibody transiently expressed in 293-F cells. Results 18 and 87 antibody failed to be expressed, G10, 26, B5 obtained expression. Specific identification results found the G10 lost the binding ability of Aβ1-42, 26, B5, and Aβ1-42-specific binding, we obtained 26, and B5 two specific whole antibodies. Finally, we pair 26 and B5 which two whole antibodies identification of epitopes, and can be combined with the AβN end (Aβ1-12, Aβ1-15) antibody affinity detection. The surface of the bacterial flagella show Aβ segment peptides of 26 and B5 epitopes identified, the results show that 26 and Aβ31-42-specific binding, B5 and Aβ1-12-specific binding and therefore initially considered 26 epitopes are located Aβ31-42, B5 epitope is located in Aβ1-12. The non-competitive ELISA results B5 affinity of KD = 1.4 × 10-8 mol / L In summary, we built a sub-Aβ1-42 epitope peptides bacterial flagellum display system, for future epitope of Aβ work and lay the foundation and success through high-throughput screening of antibody libraries from large human phage antibody library, two specific single-chain antibody 26 and B5 in the level of the whole antibody epitope identification, may be initially think 26 epitopes located within Aβ31-42, the B5 epitope is located in the Aβ1-12, measured B5 antibody affinity KD = 1.4 × 10-8 mol / L, and lay the foundation for further in-depth study of AD therapeutic antibody.

The Preparation of Human Antibodies Against Aβ

Abstract

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