Figure legends
Figure 1: A. Illustration of Ab(IgG)
purification using conjugated micellar aggregates. Target IgG + BSA are
captured by micellar aggregates comprised of a non-ionic detergent,
amphiphilic chelator (e.g. bathophenanthroline, (batho)) and
Fe2+ ions. Pure IgG is obtained by extracting IgG’s
bound to the micellar aggregates under conditions that do not co-extract
BSA or dissociate the detergent aggregates. B. Chemical
structure of the non-ionic detergents. The sum of the values represented
by the letters: W, X, Y, Z in the Tween-family is ~ 20.
The number of carbons comprising the hydrophobic anchor of: Tween-20,
40, 60 and 80, are indicated, respectively.
Figure 2: A. SDS-PAGE analysis of captured
IgG’s in pellets containing non-ionic detergents. A . Pellet
composition: Lane 1: Control: total hIgG and BSA; Lanes 2-5: Pellet
composition after hIgG capture in the presence of the Tween
representatives; Lanes 6-8: as in lanes 2-5 but in the presence of Brij
representatives; Lane 9 and 10 as in lanes 2-5 but in the presence of
Triton X-100 and Pluronic F-127, respectively. B . As inA , but with mouse IgG. BSA , H , L,A denote bovine serum albumin, the reduced h eavy
chain, the reduced l ight chain and the detergenta ggregates, respectively. C-D . Supernatant composition
after extracting IgG’s from indicated
[detergent:[(batho)3:Fe2+]
pellets with 50 mM glycine at pH 3.8 as described in Experimental.
Indicated overall process yields (i.e. IgG capture and
extraction) are shown and represent an average of 3 independent
experiments. Gels are Coomassie stained.
Figure 3: Dynamic light scattering (DLS) of detergent
aggregates. Hydrodynamic p article size of detergent aggregates
containing the non-ionic detergents and the
[(batho)3:Fe2+]-complex. The data
represent an average of at least 5 independent measurements performed on
different days.
Figure 4: Purification of IgG’s via filtration.A. Characterization of detergent aggregates for purifying hIgG
under conditions described in the Experimental section, where
centrifugation is replaced by filtration. BSA , H ,L denote bovine serum albumin, the reduced h eavy
chain, the reduced l ight chain, respectively. The asterisks
represent overall yields based on 4-6 independent experiments. Gels are
Coomassie stained. B. Dynamic light scattering (DLS) analysis
of hIgG purified via filtration with detergents as indicated.
Control - hIgG particle size (10.95 nm), not subject to detergent
aggregate purification. C. ELISA analysis. Anti-BSA IgG was
captured and extracted from indicated detergent aggregates and tested
for its ability to recognize BSA via an ELISA assay described in the
Experimental. Indicated percent values represent IgG recovery yields
relative to equal amounts of IgG not exposed to detergent aggregates
(control). Filters used were made of PVDF with a pore size of 0.1 µm
(Millipore - Ultrafree ® MC- VV).
Figure 5: Overall recovery yields with detergent
aggregates as a function of hIgG concentration. The asterisk represents
a purification protocol identical to the one described in the
Experimental section except for the following changes that were
introduced during aggregate formation: (i) FeSO4 was
replaced by FeCl2 at the same concentration. (ii) 10 mM
NaCl was increased to 200 mM NaCl.
Figure 6: Chelator recycling. Batho recyclingvia recrystallization. Yield represents an average of 5
independent experiments.
Figure 7: Light microscopy images of crystals obtained
at steps indicated in Figure 6.