Variable region differences affect antibody binding to immobilized but not soluble antigen

Abstract

We have examined the antigen binding characteristics of two chimeric IgG₁ antibodies that differ only in the heavy chain variable region. Antibodies 10B and B11 were expressed from two different anti-(Tyr, Glu)-Ala–Lys murine V_H genes joined to human IgG₁ constant region genes in a murine anti-(Tyr, Glu)-Ala-Lys heavy chain loss variant hybridoma. The binding characteristics of the antibodies to (Tyr,Glu)-Ala–Lys and to a peptide conjugate, CYYYEEEEY:BSA, were measured in solution and solid phase assays. The antibodies exhibited similar affinities and binding characteristics when assayed in solution assays. However, when we measured binding of antibodies to immobilized antigens, we found that antibody affinity depended on the epitope density in the immobilized immune complexes. The binding of antibody 10B and of B11 to immobilized (Tyr,Glu)-Ala–Lys and to CYYYEEEEY:BSA were similar at high antigen density, but antibody B11 bound less well at lower antigen density. Fab fragments of 10B bound to immobilized (Tyr,Glu)-Ala–Lys and CYYYEEEEY:BSA, but Fab fragments of B11 did not bind to (Tyr,Glu)-Ala–Lys and bound less well to CYYYEEEEY:BSA than 10B Fabs.