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 rabbit polyclonal anti-fluorescein antibodies were further resolved into subfractions based on differences in time-dependent dissociation from an FITC-adsorbent in the presence of 0.1 M fluorescein at 4 degrees C. Fab fragments separated into subpopulations based on specific dissociation times of 0.1 day, 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 determine total protein concentration of specific anti-fluorescein Fab fragments. 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 fluorescence of the bound ligand by 90% or greater. None of the plots of protein concentration versus percent yield of the total specific antibody obtained for each of the five resolved fractions constituting a specific polyclonal population conformed to Gaussian distributions. All resolved Fab subpopulations retained bound fluorescein ligand that exhibited significant bathochromic shifts in absorbancy. Based on the extent of the red-shift the antibodies segregated into one of two general spectral families showing either a peak shift to 505-507 nm or to 518-520 nm. The red-shift to 518-520 nm appeared unique to rabbit anti-fluorescein antibodies, since corresponding large shifts have not been observed with antibodies derived from other species (e.g. mouse, rat, chicken, etc.). K(d) values determined for the resolved fractions confirmed a continuous progression in affinity from the 0.1day through the 100 days elution. Preliminary isoelectric focusing analyses revealed progressive selection for relatively more homogeneous fractions, especially in the 100 days resolved fraction.
Voss
E W
EW
Department of Microbiology, University of Illinois, B103 Chemical and Life Sciences Laboratory, 607 South Goodwin Avenue, Urbana, IL 61801, USA. e-voss1@life.uiuc.edu
Croney
J C
JC
Jameson
D M
DM
D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D005591:Chemical Fractionation; D005260:Female; D019793:Fluorescein; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007525:Isoelectric Focusing; D008024:Ligands; D011817:Rabbits; D013997:Time Factors
D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D019793:Fluorescein