Binding of immunoglobulin molecules to preadsorbed protein A layers as observed by surface forces measurements

Abstract

pH-dependent interactions of immunoglobulin (IgG) to preadsorbed protein A layers were investigated by surface forces measurements. The layer of protein A preadsorbed on mica was brought into contact with monochronal IgG molecules in a 1 mM phosphate buffer containing 100 mM NaCl at pH 7.4. The IgG–protein A binding (dissociation) was detected by an increase (decrease) in the adsorbed layer thickness, as well as a concomitant outward (inward) shift in the force curve which are superimposed on accompanying conformational changes of the bottom rPA layers. At pH 7.4, the outward shift remains unaltered even after an extensive rinse with a copious amount of the pure phosphate buffer. Once the medium pH was decreased to pH 3.1, however, the force curve returned immediately to those between the pure protein A layers and the increased layer thickness disappeared. This is in accord with the pH-dependent binding and elution of IgG observed in IgG–protein A affinity chromatography. From the force data, possible conformations of adsorbed protein A and IgG–protein A complex were discussed.