DETERMINATION BY CHROMATOGRAPHIC METHODS OF THE ADSORPTION RATE-CONSTANT OF HSA ON IMMOBILIZED POLYCLONAL AND MONOCLONAL-ANTIBODIES

High performance liquid chromatography (HPLC) was used to determine th e association rate constants of antigen-antibody reactions at the soli d-liquid interface. The adsorption of human serum albumin (HSA) on mon oclonal and polyclonal anti-HSA antibodies immobilized on a HPLC silic a support was studied by frontal analysis, The theoretical model assum es irreversible adsorption during the time scale of the experiment, Th e shape of the breakthrough curves reveals the presence of several kin etic adsorption steps: the model fits well the first part of the eluti on profile. Frontal analysis gives the apparent adsorption rate consta nt, which includes the mass transfer in the stagnant fluid phase, This effect can be accounted for by carrying out studies as a function of flow rate on columns packed with adsorbents having various densities o f immobilized antibody, Within experimental error, the effective rate constant for the irreversible adsorption process is in good agreement with previous experiments performed by saturating the column with repe ated pulse injections. (C) 1995 Academic Press, Inc.