Mutational analysis of avidity and fine specificity of anti-levan antibodies

Using the polyfructose, bacterial levan, as a model polysaccharide, we anal yzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CB A/Ca mice. The BALB/c mAb were mostly germline V(H)J606:V(kappa)11, and a s ubset contained presumed somatic mutations in the complementarity-determini ng regions (CDRs) that correlated with increases in avidity for the beta(2- ->1) inulin linkage of levan, The CBA/Ca mAb were more heterogeneous in V g ene usage, but a subset of inulin-nonreactive mAb were V(H)J60G:V-A and had V-H sequence differences in the CDRs from the V(H)J606 regions of the BALB /c mAb, In this report, V(H)J606 Abs containing various combinations of spe cifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutat ions seen in CDRs of the BALB/c hybridomas were shown to directly cause mar ked increases in avidity for inulin (V-H N53H, 9-fold; V-L N53I, 20-fold; t ogether, 46-fold) but not for beta(2-->6) levan, Exchange of either positio ns 50 or 53 in V-H or the H3 loop between the BALB/c and CBA/Ca mAb resulte d in either fine specificity shift or total loss of bacterial levan binding , Three-dimensional models of the V regions suggested that residues that af fect binding to inulin alone are near the edge of the CDR surface, while re sidues involved with binding both forms of levan and affecting fine specifi city are in the V-H:V-L junctional area.