title: Considerations in producing preferentially reduced half-antibody fragments.
abstract: Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.
authors.affiliation: Department of Analytical and Environmental Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko st. 24, LT-03225 Vilnius, Lithuania. Electronic address: asta.makaraviciute@gmail.com.
authors.forename: Asta
authors.initials: A
authors.lastname: Makaraviciute
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, Garstang Building, University of Leeds, LS2 9JT Leeds, United Kingdom. Electronic address: C.D.Jackson@leeds.ac.uk.
authors.forename: Carolyn D
authors.initials: CD
authors.lastname: Jackson
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, Garstang Building, University of Leeds, LS2 9JT Leeds, United Kingdom. Electronic address: P.A.Millner@leeds.ac.uk.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Millner
authors.affiliation: Department of Analytical and Environmental Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko st. 24, LT-03225 Vilnius, Lithuania; Department of Immunology, State Research Institute Center for Innovative Medicine, Santariskiu st. 5, LT-08406 Vilnius, Lithuania. Electronic address: Almira.Ramanaviciene@chf.vu.lt.
authors.forename: Almira
authors.initials: A
authors.lastname: Ramanaviciene
mesh_terms: D000818:Animals; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D011817:Rabbits; D012756:Sheep
chemical_list: D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G
keywords: 2-mercaptoethylamine; Half-antibody fragments; Hinge region thiols; Reduced antibody; Tris(2-carboxyethyl)phosphine