Markiv, Anatoliy and Anani, Bernard and Durvasula, Ravi V. and Kang, Angray S. (2011) Module based antibody engineering: a novel synthetic REDantibody. Journal of Immunological Methods, 364 (1-2). pp. 40-49. ISSN 0022-1759
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Official URL: http://dx.doi.org/10.1016/j.jim.2010.10.009
We describe the facile generation of a stable recombinant antibody with intrinsic red fluorescent properties for qualitative and potentially quantitative immunofluorescence analysis. The REDantibody based on the X-ray crystallographic structures of the anti-sialyl-Tn antibody B72.3 and 3D model of the monomeric red fluorescent protein was designed to retain optimal spatial geometry between the C- and N- termini of the V(H) and V(L) chains respectively to mimic the domains interface pairing in antibody Fab fragments and to incorporate the red fluorescent protein as a bridging scaffold. The model was further validated by assembling a REDantibody based on CA19.9 the anti-sialylated Lewis (Le)(a) blood group antigen and 4D5-8 the anti-p185(HER2) antibodies. The chimeric heavy and light chains containing red fluorescent protein as a bridge were correctly processed and secreted into E. coli periplasm for assembly and disulphide bond formation, further analysis revealed the molecules to be exclusively monomers. Purified anti-glycan proteins were used for an immunofluorescent analysis of Trypanosoma cruzi epimastigotes, and the anti-p185(HER2) used to determine the binding properties. The REDantibody platform facilitates rapid generation of scFv chimeras that could be used for screening antibodies against cell surface makers. Furthermore, such modular assembly should permit the interchange of binding sites and of fluorophores to create robust panels of coloured antibodies.
|Research Community:||University of Westminster > Life Sciences, School of|
|Deposited On:||11 Nov 2010 14:36|
|Last Modified:||18 Oct 2012 11:38|
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