The purification of antimorphine antibodies by affinity chromatography

Abstract

Affinity chromatography was employed successfully to purify extensively antimorphine antibodies produced in rabbits. Morphine succinylated at position 6 was attached to bovine serum albumin to make it immunogenic and to Sepharose beads for affinity chromatography. A crude gamma-globulin fraction of antimorphine serum, prepared by salt fractionation, was used for purification. After unbound gamma-globulin was washed off the column with phosphate-buffered saline, two populations of antimorphine antibodies were eluted in succession by two low pH buffers. Each of these two purified antibody fractions had an index of heterogeneity of approximately 1. Immunoelectrophoresis indicated that the antibodies were immunoglobulin G and that they formed a single precipitation band with antirabbit serum. The average association constants for morphine of the two purified antibody fractions were similar and ranged from 4.5 to 4.8 X 10(7) LITERS/MOL-1. A 67-fold purification of the antimorphine antibodies could be achieved by a single pass through an affinity column.