The heterogeneity of bovine IgG2--III. The ion-exchange heterogeneity of IgG2a is the result of VH-region variation.
Bovine IgG2a from an animal homozygous at the A-locus could be fractionated on DEAE columns into subpopulations; studies using analytical size columns showed that the number of subpopulations was dependent on the ionic strength of equilibration buffer. Subpopulations of IgG2a could be purified by this method which did not differ in their carbohydrate content but had mean Ips consistent with their elution behavior. The spectrotype of their H-chains but not their L-chains, paralleled the spectrotype of the corresponding intact IgG2a subpopulations. When papain digests of each subpopulation were fractionated by chromatofocusing, their Fc fragments eluted homogeneously with a pI ca 6.0 while their Fabs eluted heterogeneously with pIs ranging from 8 to 5. The distribution of carbohydrate among such fractions corresponded to the distribution of the Fcs. The behavior of the IgG2a Fabs on chromatofocusing paralleled the elution behavior of the parent IgG2a subpopulations from DEAE-Sephadex and the isoelectric behavior of their H-chains. The antibody activity of Fab fragments, separated by chromatofocusing, are consistent with the concept of idiotypic variation. The differential antibody activity of each intact IgG2a subpopulation for seven different antigens suggests that the ion-exchange behavior of IgG2a, in which the influence of allotypes and sub-isotypes has been excluded and for which charge-related L-chain heterogeneity is minimal, must reside in the VH-regions of the different IgG2a subpopulations. Investigators are cautioned against assigning ruminant subclass designations on the basis of subpopulations isolated by ion-exchange chromatography.
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000145:classification*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D007525:Isoelectric Focusing; D010206:Papain / Q000494:pharmacology
D002241:Carbohydrates; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010206:Papain