Efficient Conjugation of Rabbit Fab′ with β‐D‐Galactosidase from Escherichia coli

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Abstract
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.