A Micro-Scale Method for the Conjugation of Affinity-Purified Fab' to β-D-Galactosidase from Escherichia coli

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Abstract
A micro-scale method for the conjugation of affinity-purified Fab′ to β-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab′)2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab′)2 was mixed with non-specific goat F(ab′)2 (0.5 mg) as a carrier, reduced with 2-mer-captoethylamine to split F(ab′)2 to Fab′ and conjugated to β-D-galactosidase using N,N′-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab′-β-D-galactosidase conjugate was separated from non-specific goat Fab′-β-D-galactosidase conjugate and unconjugated β-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 urea. The amount of the affinity-purified conjugate obtained was 56–69 μg. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.

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