Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

Abstract

a b s t r a c tRecombinant antibodies can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa antigen. Three fusion phages which bound specifically to the antigen were selected, each of which produced low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an attempt to improve its binding. Firstly, a mutant sublibrary was created by random mutagenesis. High stringency panning of this sublibrary yielded binders which produced ELISA signals up to eleven times higher than the parent scFv. An increase in the intrinsic affinity was confirmed by surface plasmon resonance. Secondly, the flexible linker between the heavy and light chains of the parent scFv was either shortened to one glycine residue or deleted entirely. No ELISA signal was obtained when the linker was absent, but the glycine-linked scFv showed enhanced binding. Size exclusion chromatography revealed that the enhanced binder had aggregated to form tetramers. This study confirms that the strategies used to improve the binding of human and mouse scFvs can also enhance chicken scFvs.

Diagnostic and Epitope Mapping Potential of Single-Chain Antibody Fragments Against Foot-and-Mouth Disease Virus Serotypes A, SAT1, and SAT3

“…Also, the monomeric scFv fragments can have moderate binding affinities when binding to a large multivalent antigen like FMDV, which is in contrast to greater binding affinities that can be achieved by the multivalent display on the phage ( 72 – 74 ). To overcome the low SAT3 scFv ELISA signals, random mutations can be introduced in the gene coding for the scFv and the length of the linker within the scFv, increasing the bacterial expression of the scFvs and thus increasing the ELISA signal ( 41 ). Another approach is to stabilize the scFv to other proteins whilst retaining functionality.…”

“…The Nkuku R phage-display library, which is a large semisynthetic library of recombinant filamentous bacteriophages displaying scFv's derived from combinatorial pairings of chicken variable heavy and light chains, was used for this study (36). This naïve library has been utilized to generate a variety of antibodies against antigens such as the bluetongue virus, African horse sickness virus, echovirus 1, coxsackievirus B3, FMDV of the SAT2 serotype as well as a mycobacterial 16 kDa antigen (25,36,(39)(40)(41)(42). These studies prove that this library is sufficiently diverse for the recognition of a variety of different haptens, proteins, bacteria, and viruses.…”

A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

“…Earlier, selection of several mouse single-chain fragments (scFvs) reactive with M. tuberculosis culture proteins by phage display was reported [23], and very recently also chicken scFv antibodies for 16 kDa hsp [24]. In the present work, VHH was preferred over scFv, because scFvs are larger, and often suffer from poor stability, unless genetically engineered [25], [26], while VHHs often display high stability over long periods of exposure to ambient temperature [10], which allowed for extensive re-use of the SPR chip.…”

Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display

“…In order to generate antibodies with the desired‐binding properties, phage‐display has been used to select antibodies on proteins or polysaccharides of Chlamydophila psittaci , Chlamydia trachomatis , Haemophilus influenzae , Listeria monocytogenes , Mycobaterium bovis , Mycobacterium tuberculosis , Porphyromonas gingivalis , Ralstonia solanacearum , Salmonella Typhimurium , and Yersinia pestis . But even selections on cell lysate or whole cells were performed on Mycobacterium avium , Bacillus anthracis , Moraxella catarrhalis , Lawsonia intracellularis , Lactobacillus acidophilus , Helicobacter pylori , Brucella melitensis , and Bordetella pertussis .…”