Resolution of rabbit polyclonal anti-fluorescein Fab (IgG) fragments into subpopulations differing in affinity and spectral properties of bound ligand

Fab fragments derived from ten different IgG populations of hyperimmune rab bit polyclonal anti-fluorescein antibodies were further resolved into subfr actions based on differences in time-dependent dissociation from an FITC-ad sorbent in the presence of 0.1 M fluorescein at 4 degreesC. Fab fragments s eparated into subpopulations based on specific dissociation times of 0.1 da y, 1.0 day, 10 days and 100 days from the adsorbent. Finally, after the 100 days elution step incubation with 6.0 M guanidine-HCl was included to dete rmine total protein concentration of specific anti-fluorescein Fab fragment s. Yields of specifically eluted Fab fragments ranged from 12.7 to 84.1% of the total Fab population originally incubated with the adsorbent. All Fab polyclonal populations and subpopulations analyzed quenched the fluorescenc e of the bound ligand by 90% or greater. None of the plots of protein conce ntration versus percent yield of the total specific antibody obtained for e ach of the five resolved fractions constituting a specific polyclonal popul ation conformed to Gaussian distributions. All resolved Fab subpopulations retained bound fluorescein ligand that exhibited significant bathochromic s hifts in absorbancy. Based on the extent of the red-shift the antibodies se gregated into one of two general spectral families showing either a peak sh ift to 505-507 nm. or to 518-520 nm. The red-shift to 518-520 am appeared u nique to rabbit anti-fluorescein antibodies, since corresponding large shif ts have not been observed with antibodies derived from other species (e.g. mouse, rat, chicken, etc.). K-d values determined for the resolved fraction s confirmed a continuous progression in affinity from the 0.1 day through t he 100 days elution. Preliminary isoelectric focusing analyses revealed pro gressive selection for relatively more homogeneous fractions, especially in the 100 days resolved fraction. (C) 2001 Published by Elsevier Science Ltd .