TY - BOOK
T1 - Betabodies and ScFv Antibodies towards Single Bead Encoding in Multiplex Diagnostics of Inflammatory Diseases
AU - Hu, Hongxia
PY - 2018
Y1 - 2018
N2 - Polymer beads of high quality with narrow size distributions were synthesised successfully withsustainable flow system, by using zero-gravity conditions and radical suspension polymerization withsilicon oil as the suspending medium. The technique was applied for the synthesis of PEGA1900 resins,optically encoded with fluorescent microparticles. The encoded beads can be applied forcombinatorial chemistry and multiplex diagnostic when combined with Ecodia technology.Single-chain variable fragment (scFv) against interleukin 1β was expressed and purified as inclusionbodies. Different methods were applied to refold the protein under various conditions. Natively foldedscFv was obtained through drop-wise addition of denatured protein to refolding buffer. scFv linkedto PEGA beads could also be refolded under different conditions, as confirmed by binding withfluorescently tagged interleukin 1β. Moreover the results showed that PEGA beads-immobilizationcan help scFv to refold to some extent. Due to the difficulty of refolding scFv β-bodies with a β hairpin structure stabilised by alternatingthreonines, were designed, to target proteins of interest specifically, according to intermolecularinteractions with the programme Molecular Operating Environment (MOE™). It has beenexperimentally confirmed that β-bodies exhibiting high binding affinity towards GFP and interleukin1β can be generated. The high selectivity of β-bodies against interleukin 1β over a closely relatedprotein interleukin 1α was demonstrated. Furthermore, GFP could be isolated from cell lysate byusing PEGA resins coupled with the relevant β-bodies, which indicates the good selectivity andbinding affinity of the designed β-body. A sandwich assay was prepared to detect small amounts of protein such as interleukin 1β, interleukin2, and interleukin 6, wherein each protein was sandwiched by two β-bodies, binding to different sitesof the target protein. Sandwich assays are very useful in providing dual specificity of the alreadyspecific β-body towards the target protein. Given the fact that β-bodies can be designed against any protein, the concept is believed to hold greatpromise in a wide range of applications such as molecular diagnostics, protein purification and drugdiscovery.
AB - Polymer beads of high quality with narrow size distributions were synthesised successfully withsustainable flow system, by using zero-gravity conditions and radical suspension polymerization withsilicon oil as the suspending medium. The technique was applied for the synthesis of PEGA1900 resins,optically encoded with fluorescent microparticles. The encoded beads can be applied forcombinatorial chemistry and multiplex diagnostic when combined with Ecodia technology.Single-chain variable fragment (scFv) against interleukin 1β was expressed and purified as inclusionbodies. Different methods were applied to refold the protein under various conditions. Natively foldedscFv was obtained through drop-wise addition of denatured protein to refolding buffer. scFv linkedto PEGA beads could also be refolded under different conditions, as confirmed by binding withfluorescently tagged interleukin 1β. Moreover the results showed that PEGA beads-immobilizationcan help scFv to refold to some extent. Due to the difficulty of refolding scFv β-bodies with a β hairpin structure stabilised by alternatingthreonines, were designed, to target proteins of interest specifically, according to intermolecularinteractions with the programme Molecular Operating Environment (MOE™). It has beenexperimentally confirmed that β-bodies exhibiting high binding affinity towards GFP and interleukin1β can be generated. The high selectivity of β-bodies against interleukin 1β over a closely relatedprotein interleukin 1α was demonstrated. Furthermore, GFP could be isolated from cell lysate byusing PEGA resins coupled with the relevant β-bodies, which indicates the good selectivity andbinding affinity of the designed β-body. A sandwich assay was prepared to detect small amounts of protein such as interleukin 1β, interleukin2, and interleukin 6, wherein each protein was sandwiched by two β-bodies, binding to different sitesof the target protein. Sandwich assays are very useful in providing dual specificity of the alreadyspecific β-body towards the target protein. Given the fact that β-bodies can be designed against any protein, the concept is believed to hold greatpromise in a wide range of applications such as molecular diagnostics, protein purification and drugdiscovery.
M3 - Ph.D. thesis
BT - Betabodies and ScFv Antibodies towards Single Bead Encoding in Multiplex Diagnostics of Inflammatory Diseases
PB - Department of Chemistry, Faculty of Science, University of Copenhagen
ER -