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- W39215364 abstract "This chapter describes a method for determination of biotin and biotin metabolites. Biotin and biotin metabolites in a sample to be assayed must first be separated by high-performance liquid chromatography (HPLC). In first incubation, avidin linked to horseradish peroxidase (HRP-avidin) is incubated with the HPLC fractions containing biotin or a biotin metabolite. The biotin or biotin metabolite binds to HRP-avidin occupying a portion of the total biotin-binding sites. During second incubation, the HRP-avidin molecules that have unoccupied biotin-binding sites bind to the biotinyl–bovine serum albumin (BSA). After second incubation, the free HRP-avidin molecules are washed away. In third incubation, the amount of HRP-avidin bound to the plate is quantitated by measuring the rate of peroxidation of the indicator dye o-phenylenediamine (OPD). The peroxidation is stopped with the addition of acid, which also intensifies the absorbance of the peroxidized OPD. Optical density (OD) at 490 nm is then quantitated in a multiwell format spectrophotometer. As increasing amounts of biotin in the standards or unknowns occupy an increasing proportion of the biotin-binding sites on HRP-avidin in the first incubation, progressively fewer molecules of HRP-avidin bind to the well in the second incubation, and color development in the third incubation is progressively less." @default.
- W39215364 created "2016-06-24" @default.
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- W39215364 date "1997-01-01" @default.
- W39215364 modified "2023-10-10" @default.
- W39215364 title "[28] Determinations of biotin in biological fluids" @default.
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- W39215364 doi "https://doi.org/10.1016/s0076-6879(97)79030-x" @default.
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