Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay.
A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.
D019297:2,4-Dinitrophenol; D000937:Antigen-Antibody Reactions; D002021:Buffers; D002846:Chromatography, Affinity; D004140:Dinitrophenols; D004926:Escherichia coli / Q000201:enzymology; D005696:Galactosidases / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000302:isolation & purification; D008301:Maleimides; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D002021:Buffers; D004140:Dinitrophenols; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008301:Maleimides; D005696:Galactosidases; D001616:beta-Galactosidase; D019297:2,4-Dinitrophenol; C023860:N,N'-2-phenylenedimaleimide