Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.