Separation of ferritin labeled antibody from free antibody

A procedure for the separation of ferritin-antibody conjugates from free antibody has been developed using isokinetic sucrose density gradient sedimentation. The gradient (5% w/w sucrose at the top to about 35% at the tube bottom) provides optimal separation of 7S free immunoglobulin from the ferritin (65S) and ferritin-antibody conjugate. Recovery of the conjugate band is simple and avoids problems of aggregation found by centrifugal pelleting and resuspension procedures. The method was tested by labeling human globulin and fractionating the gradient. Free antibody was detected at the top one-fifth of the gradient by immunodiffusion. Ferritin and the labeled antibody were found one-third to one-half of the way down the gradient. Immunoelectrophoresis was also used to demonstrate the separation of unconjugated ferritinand ferritin labeled antibody from free antibody.