Surface-density and orientation effects on immobilized antibodies and antibody fragments.

The binding parameters of randomly immobilized protein 315 and Fv fragments, as well as site-specifically immobilized Fab' fragments, have been measured for a small hapten (MW = 341 Daltons) and a large synthetic antigen (MW = 50 kD). Immobilized Fv fragments had the highest binding capacities; hence, removing unnecessary protein domains can be beneficial for improving the total capacity of an immunosorbent. For all immunosorbents, high protein loadings led to relatively low specific activities (n values). This effect was reversible, however, as the loss of immobilized antibody upon prolonged storage partially restored the specific activity. At high loadings the specific activity of immobilized whole antibody was lower for the large antigen than for the small hapten, whereas no effect of hapten size on n was evident for either immobilized Fab' or Fv fragments. Although a fraction of immobilized antibody was inactive at the higher loadings, EPR spectroscopy revealed no significant changes in the conformation of active immobilized antibody.