Fractionation and Characterization of Polyclonal Antibodies Using Three Progressively More Chaotropic Solvents

In the previous paper we described the effect of several different solvents on the structure of antibodies and demonstrated that 0.1 M glycine, pH 2.9, 7 M urea, pH 4.0, and 6 M guanidine–HCl, pH 4.0, unfold the antibodies to different degrees. Antibodies can be refolded from all of these solvents by dialysis. Polyclonal antibodies (pAbs) are a mixture of antibodies which recognize and bind different epitopes on the same antigen, with the strength of the antigen–antibody binding varying with each subpopulation. When rabbit antisera to the extracellular domain of Her2 receptor (sHer2), derived from Chinese hamster ovary cells, was applied to an antigen column, bound pAbs were recovered with a step-wise elution of 0.1 M glycine, pH 2.9 (44% of the total recovered pAb), 7 M urea, pH 4.0 (29%), and 6 M guanidine–HCl, pH 4.0 (27%), with baseline resolution between them. Fluorescence spectra of the pAbs confirmed that the 0.1 M glycine pH 2.9 sample had near-native structure, the pAbs in 7 M urea, pH 4.0, were partially unfolded, and the pAbs in the 6 M guanidine–HCl, pH 4.0, were totally unfolded. The glycine- or urea-eluted sample was refolded by dialysis into PBS, while the guanidine–HCl-eluted sample was first dialyzed into the 7 M urea pH 4.0 buffer and then into PBS. The refolded material from glycine or urea had native-like spectra, while the spectrum of the protein refolded from 6 M guanidine–HCl was slightly perturbed. All three of these subpopulations of pAbs formed antigen–antibody complexes which could be isolated by gel-filtration chromatography, precipitated sHer2 during immunoprecipitation, and recognized sHer2 in Western blots. The guanidine–HCl-eluted material was most sensitive for Western blotting. Identical results were obtained with pAbs applied either in the batch mode or to the top of the column, indicating that antibody aggregation which may occur when applied from the top of the column is not responsible for the distribution of pAbs into different subpopulations. These results indicate that the sequential use of these three increasingly chaotropic solvents to elute antibodies results in both increased recovery of antibodies and fractionation of pAbs into subpopulations with potentially different antigen binding characteristics.