| dc.description.abstract | Dynabeads™ MyOne™ Streptavidin T1 is a versatile platform that can be used for various applications due to its flexibility. By conjugating the beads with antibodies or antigen the customers themselves have biotinylated, this product can be used to produce assays tailored to each of their specific needs. Due to its flexibility and modifiability, the conjugated beads will sometimes be tailored in larger quantities, and then stored for extended periods of time under diverse conditions.
The streptavidin is covalent bound to the beads, while the biotin bound to either antibodies or antigen creates affinity bonds with amino acids on the streptavidin.Both of these bonds are susceptible to break apart from each other, rendering the complex less suitable for further use in the more sensitive immunoassays.
It is also important to avoid any free antibodies in the supernatant as they will compete with the antibodies on the beads, and reduce the signal of the assay.
Some of Thermo Fishers customers have the conjugated beads as a part of their assay kits. This makes for a situation where they want to keep the functionality of the beads even when storing them for longer periods of time.
This project was carried out to see if there was anything the customer could change in their routine that would reduce the leaching without losing the functionality.
Dynabeads™ MyOne™ Streptavidin T1 beads were conjugated with antibodies at varying concentrations, at different temperatures, and for different lengths of time.
They were also stored in different buffer solutions, and had their excess binding sites blocked with biotin. This was all to assess how different treatments of the beads affect the shelf-life stability and accuracy of the assays.
The stability of the complex was assessed after each alteration of the treatment using immunoassay methods targeting different components of the complex that could potentially dissociate from it. Changes in the beads’ functionality were also evaluated by using the beads in an assay model which sometimes included recovery tests. MODDE, a statistical software by Sartorius, was used for analysing the impact each change implemented on the treatment of the beads had, and how those correlated with the leaching of the components and the functionality of the
immunoassay based on the beads.
By using higher concentrations of biotinylated antibodies under the conjugation, and storing the beads at pH lower than physiological, with higher concentrations of BSA added to the buffer will reduce the leaching and increase the functionality of the immunoassays by reducing the noise.
Blocking unoccupied binding seats on the streptavidin with pure biotin will also reduce the noise and the leaching, more so when higher quantities of pure biotin are added to beads.
All those treatments lead to mostly exaggerated and somewhat physically impossible results in the recovery tests. Yet. those values were quite evenly elevated, so further calibration of the assays employing modified beads would be required. | en_US |
