In vitro selection and characterisation of human anti-HIV-1 antibody fragments

Tang, J. 2011. In vitro selection and characterisation of human anti-HIV-1 antibody fragments. PhD thesis University of Westminster School of Life Sciences

TitleIn vitro selection and characterisation of human anti-HIV-1 antibody fragments
TypePhD thesis
AuthorsTang, J.
Abstract

Generation of neutralising antibodies with broad specificity would be one of the effective approaches to control HIV-1 spread. It is clear that a method that allows rapid generation of neutralising antibodies is needed. This project aims at developing a novel approach to rapidly access human anti-HIV-1 antibodies in vitro by using ribosome display and selection from DNA libraries of HIV-1 patients.

Two single-chain antibody libraries (M325 and K530) were constructed from two

HIV-1 long-term non-progressors, whose sera showed cross-neutralising activities against various HIV-1 strains across a range of clades. In each library, total RNA was extracted from blood of each donor and used to synthesise cDNA. Families of 4 κ light chains, 9 λ light chains and 8 heavy chains were generated by using RT-PCR

amplification. These fragments were then assembled with all possible combinatorial pairs to form diversified repertories in the form of VL-link-VH-partial CH.

Both libraries were subjected to ribosome display for in vitro selection of functional antibodies. Ribosome display is a cell-free technique used to generate proteins that can bind to an immobilised antigen. During this process, the translated proteins are associated with their mRNAs, enabling a simultaneous selection of functional proteins and their gene. The employment of ribosome display facilitated rapid screening of two large libraries against recombinant gp120 (generated from patient K530).

Year2011
File
Publication dates
Completed2011

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