Advanced

Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective

Järås, Kerstin LU ; Ressine, Anton LU ; Nilsson, E; Malm, Johan LU ; Marko-Varga, György LU ; Lilja, Hans LU and Laurell, Thomas LU (2007) In Analytical Chemistry 79(15). p.5817-5825
Abstract
Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay.... (More)
Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostate-specific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 mu g/mL). PSA at 50 nM (1.5,mu g/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity (R-2 > 0.99) and reproducibility (coefficient of variation <= 15%), and the detection limit could be improved to 0. 14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Chemistry
volume
79
issue
15
pages
5817 - 5825
publisher
The American Chemical Society
external identifiers
  • wos:000248437700056
  • scopus:34547814065
ISSN
1520-6882
DOI
10.1021/ac0709955
language
English
LU publication?
yes
id
2d69685d-a0a9-47ba-8512-792c0a6543a3 (old id 655471)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=17605470&dopt=Abstract
date added to LUP
2007-12-13 09:52:03
date last changed
2017-09-10 03:44:29
@article{2d69685d-a0a9-47ba-8512-792c0a6543a3,
  abstract     = {Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostate-specific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 mu g/mL). PSA at 50 nM (1.5,mu g/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity (R-2 &gt; 0.99) and reproducibility (coefficient of variation &lt;= 15%), and the detection limit could be improved to 0. 14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform.},
  author       = {Järås, Kerstin and Ressine, Anton and Nilsson, E and Malm, Johan and Marko-Varga, György and Lilja, Hans and Laurell, Thomas},
  issn         = {1520-6882},
  language     = {eng},
  number       = {15},
  pages        = {5817--5825},
  publisher    = {The American Chemical Society},
  series       = {Analytical Chemistry},
  title        = {Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective},
  url          = {http://dx.doi.org/10.1021/ac0709955},
  volume       = {79},
  year         = {2007},
}