Purification of amatoxin-specific antibodies from rabbit sera by affinity chromatography, their characterization and...

Rabbits were immunized with fetuin-beta-amanitin. They produced amatoxin-specific immunoglobulins of various classes, predominantly IgG. The crude IgG fraction was isolated by gel filtration on Sephacryl S-300. By polyethylene glycol precipitation in the presence of tritiated amatoxin, as well as by a solid phase radioimmunoassay technique, the portion of amatoxin-specific antibodies in the IgG fraction was determined to be 5-13%. The affinity of the amatoxin-specific IgG for a tritiated amatoxin derivative was measured by equilibrium dialysis and calculated according to the method of Scatchard. The dissociation constant was 2.6 X 10(-9) M. The amatoxin-specific immunoglobulins were extracted by their affinity to alpha-amanitin-Sepharose 4B and eluted with excess alpha-amanitin. The complex was isolated in 95% yield with a ratio immunoglobulin:toxin of c. 2:1. Alternatively, the uncomplexed IgG could be eluted from the alpha-amanitin-Sepharose 4B with 5 M guanidine hydrochloride; this treatment, however, decreased the binding capacity for amatoxin by 30% (ratio 1.4:1). The purified amatoxin-specific IgG was assayed for its therapeutic efficiency in mice poisoned with alpha-amanitin, but was unable to neutralize the toxic effects of the mushroom toxin. On the contrary, equimolar amounts of the amatoxin-specific immunoglobulins enhanced the toxicity of alpha-amanitin in the mouse by a factor 2.