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Efficient Conjugation of Rabbit Fab′ with β‐D‐Galactosidase from Escherichia coli
Author(s) -
YOSHITAKE S.,
HAMAGUCHI Y.,
ISHIKAWA E.
Publication year - 1979
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.1365-3083.1979.tb01338.x
Subject(s) - maleimide , conjugate , chemistry , conjugated system , thiol , enzyme , escherichia coli , molecule , stereochemistry , biochemistry , polymer chemistry , organic chemistry , polymer , mathematical analysis , mathematics , gene
An efficient procedure for the conjugation of rabbit Fab′with β‐D‐galaclosidase from Escherichia coli using N,N′‐o ‐phenylenedimaleimide is described. Thiol groups of Fab′were stabilized by the presence of ethylenediaminetetraacetate, and maleimide groups were shown to be stable at pH 5 at 4°C. The stability of thiol and maleimide groups enabled an efficient introduction of maleimide groups into Fab′and the average number of maleimide groups introduced into Fab′was 0.76 (range 0.73–0.79; n =10) per molecule. As a result. 43.4% (range 41.3–46.9%; n =6) of Fab′used could be conjugated with most of β‐D‐galactosidase used. The average number of Fab′molecules conjugated per enzyme molecule was calculated to be 4.2 (range 4.0–4.6; n =6). Both the enzyme and antibody activities were well preserved in the conjugate, There was no self‐coupling of Fab′, although the enzyme was polymerized to some extent during the conjugation reaction. The enzyme activity and cross‐link in the conjugate was stable at pH 6.0–7.0 at 4°C for at least 3 months.