Advanced

Miniaturised and Quantitative Techniques Targeting Prostate Cancer

Järås, Kerstin LU (2010) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2010:73.
Abstract
The prostate-specific antigen (PSA) test used in the clinic today has been subjected to suspicions regarding specificity and sensitivity, since cases of benign prostatic hyperplasia or prostatitis are hard to distinguish from those with prostate cancer. This thesis is based upon five original papers describing efforts to develop antibody microarrays enabling improved diagnostic prostate cancer analysis. All assay developments were performed on our in-house developed and microstructured silicon surfaces, which generate increased spot signals and rapid analysis. On these surfaces, two common techniques, sandwich and reverse phase antibody microarrays were compared from a clinical perspective by analysing PSA. Using FITC-labeled detection we... (More)
The prostate-specific antigen (PSA) test used in the clinic today has been subjected to suspicions regarding specificity and sensitivity, since cases of benign prostatic hyperplasia or prostatitis are hard to distinguish from those with prostate cancer. This thesis is based upon five original papers describing efforts to develop antibody microarrays enabling improved diagnostic prostate cancer analysis. All assay developments were performed on our in-house developed and microstructured silicon surfaces, which generate increased spot signals and rapid analysis. On these surfaces, two common techniques, sandwich and reverse phase antibody microarrays were compared from a clinical perspective by analysing PSA. Using FITC-labeled detection we found the sandwich format to outperform the reversed phase assay; the sandwich assay gave five orders of magnitude better limit of detection. In an attempt to also address low abundant markers in blood serum or plasma, signal amplification was developed using europium-incorporated nanoparticles coated by streptavidin. The nanoparticles generated a signal amplification of one order of magnitude as compared to europium-labeled streptavidin. Furthermore, we validated our antibody microarray platform using 80 blood plasma samples from the clinical routine; we developed a true quantitative sandwich antibody microarray for PSA analysis in crude EDTA-plasma samples, and benchmarked the assay to a well-documented DELFIA. The first steps to a duplex assay were also taken by analysing total and free PSA on the same chip. In addition, proof-of-principle for a future lab-on-a-chip approach was shown by combining plasmapheresesis, and subsequent PSA analysis by a sandwich antibody microarray assay. The final work describes a nanovial array for rapid protein digestion and Maldi-TOF analysis; two prostate cancer biomarkers were successfully identified after 30 seconds of tryptic digestion. In conclusion, we have now developed protein chip technology to better suite the clinic within the field of prostate cancer diagnostics. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • LaBaer, Joshua, Biodesign Institute at Arizona State University, Center for Personalized Diagnostics, Tempe, AZ, USA
organization
publishing date
type
Thesis
publication status
published
subject
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2010:73
pages
158 pages
publisher
Department of Laboratory Medicine, Lund University
defense location
E1406, Ehuset, LTH
defense date
2010-06-17 09:00
ISSN
1652-8220
ISBN
978-91-86443-89-4
language
English
LU publication?
yes
id
595a8628-6154-4e27-ac09-d053bef36f4c (old id 1625002)
date added to LUP
2010-06-29 08:58:21
date last changed
2018-05-29 10:14:55
@phdthesis{595a8628-6154-4e27-ac09-d053bef36f4c,
  abstract     = {The prostate-specific antigen (PSA) test used in the clinic today has been subjected to suspicions regarding specificity and sensitivity, since cases of benign prostatic hyperplasia or prostatitis are hard to distinguish from those with prostate cancer. This thesis is based upon five original papers describing efforts to develop antibody microarrays enabling improved diagnostic prostate cancer analysis. All assay developments were performed on our in-house developed and microstructured silicon surfaces, which generate increased spot signals and rapid analysis. On these surfaces, two common techniques, sandwich and reverse phase antibody microarrays were compared from a clinical perspective by analysing PSA. Using FITC-labeled detection we found the sandwich format to outperform the reversed phase assay; the sandwich assay gave five orders of magnitude better limit of detection. In an attempt to also address low abundant markers in blood serum or plasma, signal amplification was developed using europium-incorporated nanoparticles coated by streptavidin. The nanoparticles generated a signal amplification of one order of magnitude as compared to europium-labeled streptavidin. Furthermore, we validated our antibody microarray platform using 80 blood plasma samples from the clinical routine; we developed a true quantitative sandwich antibody microarray for PSA analysis in crude EDTA-plasma samples, and benchmarked the assay to a well-documented DELFIA. The first steps to a duplex assay were also taken by analysing total and free PSA on the same chip. In addition, proof-of-principle for a future lab-on-a-chip approach was shown by combining plasmapheresesis, and subsequent PSA analysis by a sandwich antibody microarray assay. The final work describes a nanovial array for rapid protein digestion and Maldi-TOF analysis; two prostate cancer biomarkers were successfully identified after 30 seconds of tryptic digestion. In conclusion, we have now developed protein chip technology to better suite the clinic within the field of prostate cancer diagnostics.},
  author       = {Järås, Kerstin},
  isbn         = {978-91-86443-89-4},
  issn         = {1652-8220},
  language     = {eng},
  pages        = {158},
  publisher    = {Department of Laboratory Medicine, Lund University},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Miniaturised and Quantitative Techniques Targeting Prostate Cancer},
  volume       = {2010:73},
  year         = {2010},
}