Elimination of Nonspecific Binding to Plasma Membranes in Indirect Immunoferritin Technique

Reduction of nonspecific binding of ferritin conjugated to anti-rabbit IgG goat IgG with the aid of p, p'-difluoro-m, m'-dinitro diphenyl sulfone was investigated using the plasma membranes of guinea pig poly-morphonuclear leukocytes as antigen. The ferritin conjugate was labeled with 125I and fractionated by DEAE cellulose column chromatography into four fractions. Each fraction was incubated with the plasma membrane fractions, and fraction 2 eluted with a buffer of low ionic strength gave the least radio-activity bound to plasma membrane without specific antibody against the membrane (nonspecific binding). On further absorption of this fraction with plasma membrane, highly specific ferritin conjugate which showed only 1.2% of the nonspecific binding of unfractionated ferritin conjugate was recovered in the non-adsorbed fraction.