Limits...
Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding.

Scott N, Qazi O, Wright MJ, Fairweather NF, Deonarain MP - Mol. Immunol. (2010)

Bottom Line: We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity.This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells.For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Exhibition Road, London, SW7 2AZ, UK.

ABSTRACT
An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.

Show MeSH

Related in: MedlinePlus

Bst NI fingerprint analyses of scFv clones. Bst NI RFLP analysis of scFv clones isolated from a phagemid library panned against TeNT-Hc and verified as target specific by ELISA. The scFv genes were amplified from minipreped phagemid vector by PCR using primers LMB3 and FDSEQ1. The product was digested with Bst NI and then underwent electrophoretic separation on a 4% agarose gel stained with ethidium bromide. Marker is Hyperladder IV (Bioline). Gel A (HcC clones), Gel B (HcJ clones) and Gel C (HcN clones). Clone numbers refer to original screening designation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874839&req=5

d32e3152: Bst NI fingerprint analyses of scFv clones. Bst NI RFLP analysis of scFv clones isolated from a phagemid library panned against TeNT-Hc and verified as target specific by ELISA. The scFv genes were amplified from minipreped phagemid vector by PCR using primers LMB3 and FDSEQ1. The product was digested with Bst NI and then underwent electrophoretic separation on a 4% agarose gel stained with ethidium bromide. Marker is Hyperladder IV (Bioline). Gel A (HcC clones), Gel B (HcJ clones) and Gel C (HcN clones). Clone numbers refer to original screening designation.


Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding.

Scott N, Qazi O, Wright MJ, Fairweather NF, Deonarain MP - Mol. Immunol. (2010)

Bst NI fingerprint analyses of scFv clones. Bst NI RFLP analysis of scFv clones isolated from a phagemid library panned against TeNT-Hc and verified as target specific by ELISA. The scFv genes were amplified from minipreped phagemid vector by PCR using primers LMB3 and FDSEQ1. The product was digested with Bst NI and then underwent electrophoretic separation on a 4% agarose gel stained with ethidium bromide. Marker is Hyperladder IV (Bioline). Gel A (HcC clones), Gel B (HcJ clones) and Gel C (HcN clones). Clone numbers refer to original screening designation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874839&req=5

d32e3152: Bst NI fingerprint analyses of scFv clones. Bst NI RFLP analysis of scFv clones isolated from a phagemid library panned against TeNT-Hc and verified as target specific by ELISA. The scFv genes were amplified from minipreped phagemid vector by PCR using primers LMB3 and FDSEQ1. The product was digested with Bst NI and then underwent electrophoretic separation on a 4% agarose gel stained with ethidium bromide. Marker is Hyperladder IV (Bioline). Gel A (HcC clones), Gel B (HcJ clones) and Gel C (HcN clones). Clone numbers refer to original screening designation.
Bottom Line: We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity.This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells.For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Exhibition Road, London, SW7 2AZ, UK.

ABSTRACT
An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.

Show MeSH
Related in: MedlinePlus