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  • Title: Inadvertent BSA-induced elution of IgE in the BSA-RAST.
    Author: Kleine Budde I, de Heer PG, Stapel SO, van der Zee JS, Aalberse RC.
    Journal: Allergy; 2001 Nov; 56(11):1055-60. PubMed ID: 11703218.
    Abstract:
    BACKGROUND: Bovine serum albumin (BSA) is widely used to block nonspecific binding in immunochemical assays. Whereas a previous study had indicated that soluble allergen present during the incubation with anti-IgE in the RAST did not affect bound IgE, we reinvestigated this in the current study, using IgE elution from BSA by soluble BSA as a test system. METHODS: Sepharose-coupled BSA (0.08, 0.4, 2, or 10 microg BSA/test) was incubated overnight with serum and washed. The Sepharose was then incubated with different concentrations of soluble BSA (0, 12, 60, 300, or 1500 microg/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, and 20 h). RESULTS: Incubation in buffer without BSA did not change IgE binding. Soluble BSA eluted IgE antibodies from immobilized BSA by up to 85%. If the BSA density on the solid phase was > or =2 microg/test, the elution efficiency was dependent on the levels of both immobilized BSA and soluble BSA. At lower densities, the dissociation was dependent only on the concentration of soluble BSA. The time needed to obtain 50% IgE elution (t(1/2)) was less if the density of immobilized BSA decreased. Below the critical density (0.8 microg BSA/mg solid phase), t(1/2) was independent of the coating density (45 min). Probably all IgE antibodies are monovalently bound below this density. CONCLUSIONS: Dissociation of IgE from immobilized protein in the presence of soluble protein should be taken into account, particularly when IgE to mammalian serum albumin is involved (milk, meat, or animal dander).
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