Production and characterisation of novel recombinant antibody fragments against sialic acid
Donohoe, Gerard Gary (2011) Production and characterisation of novel recombinant antibody fragments against sialic acid. PhD thesis, Dublin City University.
Full text available as:
Sialic acids are a family of acidic monosaccharides that typically reside as terminal moieties on N- and O-linked glycans. These sugars are actively involved in a plethora of biological phenomena, ranging from cell-cell adhesion and recognition, intracellular signalling events, pathogen attack, viral infection and inflammatory disease. In addition, many cancer-related antigens contain terminal sialic acids or altered sialylation patterns. The identification of sialic acid-specific antibodies is important in the fields of basic research and diagnostics. Therefore the primary objective of this thesis was the generation and characterisation of recombinant antisialic
acid antibodies.A single-chain antibody fragment (scFv) library was constructed from a chicken that was immunised with a novel synthetic sialic acid protein-conjugate (Neu5Gchumanserum albumin). The scFv library was biopanned using a second novel sialoneoglycoconjugate(Neu5Gc-bovine serum albumin). Anti-sialic scFvs were isolated byphage-display and binding activity, multimeric status, and multivalent properties wereassessed by ELISA, HPLC, FPLC, and SPR. One clone, AE8, displayed the strongest reactivity to a panel of different sialylated structures. In addition, SPR inhibition andkinetic analysis revealed that the AE8 scFv had nanomolar affinity for the Neu5Gc-BSA neoglycoconjugate. The heavy chain gene of the AE8 scFv was assembled with a repertoire of error prone PCR-amplified light chain genes, that originated from the unpanned anti-sialic acid library. The mutant scFv library was biopanned using a depletion protocol and two ovalbumin (OVA) conjugates, Neu5Gc-linker1-OVA and linker1-OVA. ELISA analysis, SPR inhibition and ‘off-rate’ studies identified one mutant clone, E15, which displayed strong affinity for conjugates of sialic acid. A solution-phase SPR-based inhibition study demonstrated that the E15 scFv had an IC50 concentration of 1.6ng/mL for the Neu5Gc-BSA conjugate. When compared to the parental AE8 clone (IC50 of 5.7ng/mL) the mutant E15 scFv exhibited an improvement in sialic acid binding of approximately 3.5-fold. The generation of such highly specific anti-sialic acid antibodies in a recombinant format is exceedingly rare. Most anti-carbohydrate antibodies are not recombinant and display weak carbohydrate-protein interactions. This work shows for the first time, the generation and directed evolution of anti-carbohydrate scFvs that have nanomolar affinity for Neu5Gc-containing structures.
Archive Staff Only: edit this record