Binding studies on isoelectrofocused fractions from a DNP antibody population.

Preparative liquid isoelectric focusing has been used to isolate antibody fractions of restricted heterogeneity from typically heterogeneous anti-DNP antibody populations. The focusing distributions of anti-DNP antibody isolated from different rabbits and from different bleed periods (separated by 120 days) of the same rabbit were qualitatively very similar. All antibody preparations contained major components focusing in the pH ranges 7.5–8.5, 6.4–6.6, 5.8–6.1, and 5.3–5.7. However, demonstrable quantitative differences in the relative proportions of the pH 6.4–6.6 component and the pH 5.3–5.7 component were seen in comparing the focusing distributions of antibody isolated from the two bleed series of the same rabbit. Three of the focused fractions from the later bleed period were refocused over appropriate pH ranges yielding components considerably restricted in charge heterogeneity. Binding parameters determined for the refocused fractions were indicative of functional homogeneity. There was no correlation between binding affinity and antibody electrical charge. Additionally, there was no consistent relationship between the spectral properties (extinction coefficients and maximum quench values) of the refocused fractions and their relative affinities. The qualitative similarities and the quantitative differences observed in the focusing distributions of antibody isolated from different bleed series of the same rabbit are consistent with a selectional theory of antibody production. The results of binding experiments suggest a somewhat anomalous role for antibody tryptophan in the binding of dinitrophenyl ligands.