Update on Biotin Therapy in Dermatology: Time for a Change.

Biotin (vitamin B7 or H) is found in milk, nuts, egg yolks, cereals, supplements, synthesized by intestinal bacteria, and is required for gluconeogenesis, fatty acid synthesis and amino acid catabolism.

FRET or no FRET: a quantitative comparison.

Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein interactions, and in biophysical projects it is used as a quantitative measure for distances between a single donor and acceptor molecule. Numerous approaches can be found in the literature to quantify and map FRET, but the measures they provide are often difficult to interpret. We propose here a quantitative comparison of these methods by using a surface FRET system with controlled amounts of donor and acceptor fluorophores and controlled distances between them. We support the system with a Monte Carlo simulation of FRET, which provides reference values for the FRET efficiency under various experimental conditions. We validate a representative set of FRET efficiencies and indices calculated from the different methods with different experimental settings. Finally, we test their sensitivity and draw conclusions for the preparation of FRET experiments in more complex and less-controlled systems.

Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.

A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement of (strept)avidin in complex, colored biofluids, such as crude egg white or serum relies on a simple titration protocol. Only occasional recalibration of the reagent solution is required. Due to these merits the proposed assay is particularly suited for rapid measurement of few samples on short notice, for functional control of (strept)avidin-containing reagents after storage, and for the monitoring of (strept)avidin concentrations in large scale processes.

Indirect enzyme-linked method for determining biotin in human serum.

An indirect enzyme-linked assay was developed for quantifying biotin concentrations in human sera. Biotin standard solutions or unknown samples are preincubated with streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and added to plates coated with biotinylated bovine IgG (B-IgGb). The concentration of the streptavidin-HRP is such that the streptavidin binding sites are sufficient to bind apparently all the biotin present in samples, whereas, the remaining sites are inversely proportional to the amount of biotin in analysed sample. These sites could subsequently interact with the immobilized B-IgGb providing signal. The assay demonstrated dynamic range 5 to 640 ng/L, detection limit 2 ng/L, intra- and interassay C.V., 1.6-3.9% and 3.7-7.2% respectively, recovery 100-114% and linear recovery 90-117%. Serum biotin determined: healthy individuals 66 to 600 ng/L, pregnant women (> or = 36 weeks) 60 to 360 ng/L, and patients under chronic haemodialysis 0.56 to 1.62 micrograms/L. The method described is among those few which have been experimentally evaluated for their capabilitity of assessing biotin in human sera.

An enzymeimmunoassay for total thyroxine using avidin-biotin separation system and thyroxine-peroxidase conjugate.

An avidin-biotin enzymeimmunoassay for total thyroxine in serum is described. Avidin was adsorbed to biotinylated bovine serum albumin coated tubes prepared with glutaraldehyde as coupling agent. In the enzymeimmunoassay, affinity purified biotinylated anti-thyroxine IgG, sample or standards, and thyroxine-horseradish peroxidase were simultaneously added to the avidin coated tubes. The bound enzymatic activity was then measured with o-phenylenediamine and H2O2. Results showed that the assay has good precision (within-assay CV% less than 10% and between assay 11.7% in hypo- and 6.9% in hyperthyroid range), good assay range (0-800 nmol/L), good sensitivity (4 nmol/L), and can be performed in 2.5 hours. The results obtained correlate well (r = 0.93) with those of an RIA.