title: Homogeneous antibody fragment conjugation by disulfide bridging introduces 'spinostics'.
abstract: A major obstacle to the efficient production of antibody conjugates for therapy and diagnosis is the non-ideal performance of commonly used chemical methods for the attachment of effector-molecules to the antibody of interest. Here we demonstrate that this limitation can be simply addressed using 3,4-substituted maleimides to bridge and thus functionalize disulfide bonds to generate homogeneous antibody conjugates. This one-step conjugation reaction is fast, site-specific, quantitative and generates products with full binding activity, good plasma stability and the desired functional properties. Furthermore, the rigid nature of this modification by disulfide bridging enables the successful detection of antigen with a spin labeled antibody fragment by continuous-wave electron paramagnetic resonance (cw-EPR), which we report here for the first time. Antigen detection is concentration dependent, observable in human blood and allows the discrimination of fragments with different binding affinity. We envisage broad potential for antibody based in-solution diagnostic methods by EPR or 'spinostics'.
authors.affiliation: Department of Chemistry, University College London, 20 Gordon Street, London, WC1H OAJ, UK.
authors.forename: Felix F
authors.initials: FF
authors.lastname: Schumacher
authors.forename: Vishal A
authors.initials: VA
authors.lastname: Sanchania
authors.forename: Berend
authors.initials: B
authors.lastname: Tolner
authors.forename: Zoë V F
authors.initials: ZV
authors.lastname: Wright
authors.forename: Chris P
authors.initials: CP
authors.lastname: Ryan
authors.forename: Mark E B
authors.initials: ME
authors.lastname: Smith
authors.forename: John M
authors.initials: JM
authors.lastname: Ward
authors.forename: Stephen
authors.initials: S
authors.lastname: Caddick
authors.forename: Christopher W M
authors.initials: CW
authors.lastname: Kay
authors.forename: Gabriel
authors.initials: G
authors.lastname: Aeppli
authors.forename: Kerry A
authors.initials: KA
authors.lastname: Chester
authors.forename: James R
authors.initials: JR
authors.lastname: Baker
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000627:therapeutic use; D000941:Antigens / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D004220:Disulfides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008301:Maleimides / Q000737:chemistry*; D013113:Spin Labels
chemical_list: D000906:Antibodies; D000941:Antigens; D004220:Disulfides; D008301:Maleimides; D013113:Spin Labels