Enzyme-linked immunosorbent assay (ELISA)
Nunc-Immuno Microwell plates (F96 Maxisorp) were first coated with 2%
BSA (200 μL), left for overnight incubation at 4 °C and excess of BSA
was removed with three aliquots of PBS (200 μL). Purified anti-BSA IgGs,i.e ., IgGs that were captured by either Tween-20, Brij-O20 or
Triton X-100 aggregates and extracted with Gly buffer (see the
Extraction section of IgG’s from detergent aggregates), were then added
to the washed wells. Accordingly, 100 μL of purified and diluted
(1:1000) naked anti-BSA IgG (from rabbit) were added to the BSA coated
wells, incubated for 2 hours at room temperature (RT) and unbound
anti-BSA IgG’s were then removed with PBS (3 x 200 μL). To wells
containing the anti-BSA IgG, a diluted (1:15,000) anti-rabbit IgG-HRP
conjugate was introduced into wells containing the naked IgG. In both
cases, the system was further incubated for 1 hour at RT and excess HRP
conjugates (either streptavidin or IgG) were excluded with PBS (3 x 200
μL). Addition of the HRP substrate (1 X TMB solution) was followed by 10
minutes of incubation at RT and the reaction was stopped upon addition
of 2 N H2SO4 (50 μL). The intensity of
the yellow color in the wells was measured at 450 nm using an ELISA
reader (Tecan infinite M200).