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Parallel comparison of sandwich and direct label assay protocols on cytokine detection protein arrays

Publication ,  Journal Article
Li, Y; Nath, N; Reichert, WM
Published in: Analytical Chemistry
October 1, 2003

This study describes a systematic comparison of the preparation, sensitivity, and response of direct label and sandwich fluoroimmunoassay protein arrays for parallel detection of cytokines and growth factors. Five model analytes were examined, IL-1β, TNF-α, VEGF, MIP-1β, and TGF-β1, as well as four different array printing slides. Each slide was printed with four identical anti-cytokine arrays, two arrays employed for direct labeling and two for sandwich assays. All five target cytokines assayed using the sandwich format significantly outperformed the corresponding direct label assays in terms of background-subtracted fluorescent intensity, although the extent varied for different cytokines, growth factors, and slides examined. Binding of target protein by immobilized capture antibody was examined by Biacore surface plasmon resonance with and without labeling of the target protein. A major explanation for the superior performance of the sandwich assay protocol was the dilution of the labeled target cytokine in the size exclusion gel filtration column during labeling procedure. Other possible explanations included that binding of the detection antibody-induced signal amplification, and labeling of target cytokine reduced efficiency of cytokine binding to immobilized capture antibody.

Duke Scholars

Published In

Analytical Chemistry

DOI

ISSN

0003-2700

Publication Date

October 1, 2003

Volume

75

Issue

19

Start / End Page

5274 / 5281

Related Subject Headings

  • Analytical Chemistry
  • 4004 Chemical engineering
  • 3401 Analytical chemistry
  • 3205 Medical biochemistry and metabolomics
  • 0399 Other Chemical Sciences
  • 0301 Analytical Chemistry
 

Citation

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Li, Y., Nath, N., & Reichert, W. M. (2003). Parallel comparison of sandwich and direct label assay protocols on cytokine detection protein arrays. Analytical Chemistry, 75(19), 5274–5281. https://doi.org/10.1021/ac034563f
Li, Y., N. Nath, and W. M. Reichert. “Parallel comparison of sandwich and direct label assay protocols on cytokine detection protein arrays.” Analytical Chemistry 75, no. 19 (October 1, 2003): 5274–81. https://doi.org/10.1021/ac034563f.
Li Y, Nath N, Reichert WM. Parallel comparison of sandwich and direct label assay protocols on cytokine detection protein arrays. Analytical Chemistry. 2003 Oct 1;75(19):5274–81.
Li, Y., et al. “Parallel comparison of sandwich and direct label assay protocols on cytokine detection protein arrays.” Analytical Chemistry, vol. 75, no. 19, Oct. 2003, pp. 5274–81. Scopus, doi:10.1021/ac034563f.
Li Y, Nath N, Reichert WM. Parallel comparison of sandwich and direct label assay protocols on cytokine detection protein arrays. Analytical Chemistry. 2003 Oct 1;75(19):5274–5281.
Journal cover image

Published In

Analytical Chemistry

DOI

ISSN

0003-2700

Publication Date

October 1, 2003

Volume

75

Issue

19

Start / End Page

5274 / 5281

Related Subject Headings

  • Analytical Chemistry
  • 4004 Chemical engineering
  • 3401 Analytical chemistry
  • 3205 Medical biochemistry and metabolomics
  • 0399 Other Chemical Sciences
  • 0301 Analytical Chemistry