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.