A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli.
A micro-scale method for the conjugation of affinity-purified Fab' to beta-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-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. 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.
D000818:Animals; D006063:Chorionic Gonadotropin / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D005696:Galactosidases / Q000032:analysis*; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D007074:Immunoglobulin G / Q000302:isolation & purification; D011137:Polystyrenes; D011485:Protein Binding
D006063:Chorionic Gonadotropin; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011137:Polystyrenes; D005696:Galactosidases