Inadvertent BSA-induced elution of IgE in the BSA-RAST

Background: Bovine serum albumin (BSA) is widely used to block nonspecific binding in immunochemical assays. Whereas a previous study had indicated th at soluble allergen present during the incubation with anti-IgE in the RAST did not affect bound IgE, we reinvestigated this in the current study, usi ng IgE elution from BSA by soluble BSA as a test system. Methods: Sepharose-coupled BSA (0.08, 0.4, 2, or 10 mug BSA/test) was incub ated overnight with serum and washed. The Sepharose was then incubated with different concentrations of soluble BSA (0, 12, 60, 300, or 1500 mug/test) , washed again, and incubated with radioactive anti-IgE. The effect on IgE binding was investigated for various incubation periods (t = 0, 1, 2, 4, an d 20 h). Results: Incubation in buffer without BSA did not change IgE binding. Solub le BSA eluted IgE antibodies from immobilized BSA by up to 85%. If the BSA density on the solid phase was 2 mug/test, the elution efficiency was depen dent on the levels of both immobilized BSA and soluble BSA. At lower densit ies, the dissociation was dependent only on the concentration of soluble BS A. The time needed to obtain 50% IgE elution (t(1/2)) was less if the densi ty of immobilized BSA decreased. Below the critical density (0.8 mug BSA/mg solid phase), t(1/2) was independent of the coating density (45 min). Prob ably all IgE antibodies are monovalently bound below this density. Conclusions: Dissociation of IgE from immobilized protein in the presence o f soluble protein should be taken into account, particularly when IgE to ma mmalian serum albumin is involved (milk, meat, or animal dander).