Antibody elution from hapten-cellulose immunoadsorbents: the effects of hapten structure pH, and salt concentration

Hapten-celluose immunoadsorbents were prepared using two negatively charged ( p-azobenzenearsonate and p-azobenzoate), one positively charged ( p-azophenyltrimenthylammonium), one neutral hydrophilic ( p-azophenyl β- d-glucopyranoside), and one neutral more hydrophobic (2,4-dinitrophenyl) haptens. The elution properties of 131I labeled whole globulin fractions derived from rabbits immunized with edestin-hapten or human γ-globulin-hapten conjugates were studied at different pH, salt or buffer concentrations using a separate hapten-cellulose microcolumn for each eluting solution. A substantial fraction of the anti-benzenearsonate antibodies could be eluted between pH 5 anf 7 and below pH 3. Anti-benzoate antibodies were also eluted above pH 5 and below pH 3, but less antibody was eluted in the higher pH range. In contrast, significant elution of the anti-phenyltrimethyl-ammonium and anti-phenyl β- d-glucopyranoside antibodies was observed only with lower pH buffers. The anti-2,4-dinitrophenyl antibodies were poorly eluted with any pH salt combination used. Specifically purified 7S and 19S anti-benzenearsonate antibodies had elution properties increased elution in the high pH range (5–7), suggesting the prime importance of charge interactions. Added salt reduced the amount of antibody eluted in the low pH range (1–3), however, suggesting the additional factor of hydrophobic interactions. Studies with labeled benzenearsonate antibodies indicated that the differences in salt dependences were a result of differences, within the same antibody population, int he nature of the forces involved in the antigen-antibody interactions as the pH was changed.