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Formation of Covalent C3b‐Tetanus Toxin Complexes: a Tool for the In Vitro Study of Antigen Presentation
Author(s) -
VILLIERS* M.B.,
VILLIERS C. L.,
WRIGHT J. F.,
MAISON C. M.,
COLOMB M. G.
Publication year - 1991
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1991.tb01582.x
Subject(s) - chemistry , covalent bond , antigen , size exclusion chromatography , in vitro , toxin , biochemistry , chromatography , dissociation (chemistry) , cleavage (geology) , enzyme , organic chemistry , biology , immunology , paleontology , fracture (geology)
A novel method is described for the formation and purification of covalent complexes between the complement component C3b and an antigen (tetanus toxin. TT). using purified proteins in fluid phase. C3b is generated in situ by tryptic cleavage or C3 after co‐precipitation of C3 and TT in the presence of polyethylene glycol. Various parameters were analysed to optimize complex formation: under conditions which minimized the formation ofcovalentC3bmultimers, 30% and 8% respectively ofC3band TT were incorporated into covalent one‐to‐one complexes which were purified using gel filtration chromatography. The linkage WAS localized between the α chain of C3b and either the H or L chain of TT; n required the in situ formation of C3b and was partially destroyed by 1 M hydroxylamine. Spontaneous dissociation of the complex could be partly avoided by HgCl 2 , a thiol reagent which inhibits the esterase‐like activity of bound C3b, These findings suggest the involvement of the reactive carbonyl of nascent C3b with hydroxyl groups of TT, Such C3b TT complexes provide a defined tool to analyse the influence of antigen‐bound C3bon antigen addressing and intracellular processing by antigen‐presenting cells.