Abstract
The presented data refer to optimization and quantitative characterization of a rapid lateral flow assay based on high-affinity bifunctional ligand and magnetic nanolabels, which was developed for detection of small molecules of thyroid hormones. The results were obtained by several techniques, including the magnetic particle quantification method, spectral-correlation interferometry and spectral-phase interferometry, dynamic light scattering, enzyme linked immunosorbent assay. The long-term stability of “antibody – magnetic nanoparticle” conjugates is shown. The assay specificity is confirmed, and verification of successful combination of magnetic particles and antibodies is demonstrated. The kinetic and equilibrium dissociation constants are determined for interactions between thyroxine and monoclonal antibodies. The obtained data could be used for design of other platforms for detection of small molecules.
Highlights
The obtained data show optimization and validation of all stages of the developed magnetic lateral flow assay for quantitative and ultrasensitive express-detection of free thyroxine [1]
The assay was performed in an original competitive format with streptavidin on the test line (TL) of a lateral flow test strip and biotinylated thyroxin (T4-bt) competing with free thyroxin for the limited amount of antibody binding sites in sample solution
The signal from test line was measured by the Magnetic Particle Quantification (MPQ) readers described in details in [2,3]
Summary
The obtained data show optimization and validation of all stages of the developed magnetic lateral flow assay for quantitative and ultrasensitive express-detection of free thyroxine [1]. The assay was performed in an original competitive format with streptavidin on the test line (TL) of a lateral flow test strip and biotinylated thyroxin (T4-bt) competing with free thyroxin for the limited amount of. The obtained conjugates (volume 2 μl) and 12 μl of T4-bt were premixed with 80–μl samples of human serum containing known concentrations of free thyroxin and incubated during 15 min at room temperature. The lateral flow test strips with streptavidin on TL were placed vertically into the samples. The original design and quantitative optimization of each stage of the assay has permitted attractive limit of detection for free thyroxin in human serum of 20 fM or 16 fg/ml. The data below refer to assay characterization and can be used for effective design of rapid and sensitive test-systems for detection of small molecules
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