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ScFv barcoding – technology development towards MIcroArrays in Solution

Kovacic, Rebeka LU (2015) KIMM01 20142
Educational programmes
Department of Immunotechnology
Abstract
The field of disease proteomics is lacking techniques capable of analysing complex samples with high sensitivity and selectivity. A new MIcroArrays in Solution (MIAS) technology is being developed to addres the technological issues of the current established methods, such as antibody microarrays, 2DE and MS used for protein expression profiling. MIAS technology is based on DNA barcode labeled scFvs, freely floating in the complex sample mixture, which would enable us to avoid the technical and logistical challenges associated with planar antibody microarrays, but also achieve a sensitive, multiplex and quantitative assay. In this project, we used two technology approaches for oligonucleotide tagging of recombinant scFv, namely i)... (More)
The field of disease proteomics is lacking techniques capable of analysing complex samples with high sensitivity and selectivity. A new MIcroArrays in Solution (MIAS) technology is being developed to addres the technological issues of the current established methods, such as antibody microarrays, 2DE and MS used for protein expression profiling. MIAS technology is based on DNA barcode labeled scFvs, freely floating in the complex sample mixture, which would enable us to avoid the technical and logistical challenges associated with planar antibody microarrays, but also achieve a sensitive, multiplex and quantitative assay. In this project, we used two technology approaches for oligonucleotide tagging of recombinant scFv, namely i) heterobifunctional conjugation and ii) click-chemistry followed by light-dependent crosslinking using recombinant scFv equipped with a photoaffinity tag. Our results showed large reproducibility problems of heterobifunctional conjugation, requiring extensive optimization in the future. In addition, the technology was labor intensive and time consuming, making it a poor candidate for the new developing technology. However, site-specific photo crosslinking was rather straightforward, quick and performed with the basic lab equipment, making it a promising candidate for the MIAS technology. The click chemistry reaction between β-CD and oligonucleotides, though, needs further optimization and validation, and the structural and functional properties of oligonucleotide tagged scFvs need to be investigated as well. Since the site-specific coupling was performed with no major difficulties, more emphasis should be put on methods dealing with validation, detection and purification of the conjugates. (Less)
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author
Kovacic, Rebeka LU
supervisor
organization
course
KIMM01 20142
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
7761528
date added to LUP
2015-08-24 13:57:20
date last changed
2015-08-24 13:57:20
@misc{7761528,
  abstract     = {The field of disease proteomics is lacking techniques capable of analysing complex samples with high sensitivity and selectivity. A new MIcroArrays in Solution (MIAS) technology is being developed to addres the technological issues of the current established methods, such as antibody microarrays, 2DE and MS used for protein expression profiling. MIAS technology is based on DNA barcode labeled scFvs, freely floating in the complex sample mixture, which would enable us to avoid the technical and logistical challenges associated with planar antibody microarrays, but also achieve a sensitive, multiplex and quantitative assay. In this project, we used two technology approaches for oligonucleotide tagging of recombinant scFv, namely i) heterobifunctional conjugation and ii) click-chemistry followed by light-dependent crosslinking using recombinant scFv equipped with a photoaffinity tag. Our results showed large reproducibility problems of heterobifunctional conjugation, requiring extensive optimization in the future. In addition, the technology was labor intensive and time consuming, making it a poor candidate for the new developing technology. However, site-specific photo crosslinking was rather straightforward, quick and performed with the basic lab equipment, making it a promising candidate for the MIAS technology. The click chemistry reaction between β-CD and oligonucleotides, though, needs further optimization and validation, and the structural and functional properties of oligonucleotide tagged scFvs need to be investigated as well. Since the site-specific coupling was performed with no major difficulties, more emphasis should be put on methods dealing with validation, detection and purification of the conjugates.},
  author       = {Kovacic, Rebeka},
  language     = {eng},
  note         = {Student Paper},
  title        = {ScFv barcoding – technology development towards MIcroArrays in Solution},
  year         = {2015},
}