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Microformat Imaging ELISA for Pesticide Determination

Dzgoev, Anatoli ; Mecklenburg, Michael ; Larsson, Per-Olof LU and Danielsson, Bengt LU (1996) In Analytical Chemistry 68(19). p.3364-3369
Abstract
A flat-well microformat competitive enzyme-linked immunosorbent
chemiluminescent assay for the detection of the pesticide
2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film
technology was used to pattern a hydrophobic layer 100 μm thick onto
glass microscope slides to form an array of 2 × 2 mm2
squares. These flat wells were able to hold 2 μL of reagents,
corresponding to a height of ∼500 μm, with minimal contamination risk.
The hydrophobic ink used to pattern the surfaces allowed significantly
larger volumes of samples to be applied when compared with surfaces
patterned with nonhydrophobic inks. This reduced evaporation effects and
permitted greater pipetting... (More)
A flat-well microformat competitive enzyme-linked immunosorbent
chemiluminescent assay for the detection of the pesticide
2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film
technology was used to pattern a hydrophobic layer 100 μm thick onto
glass microscope slides to form an array of 2 × 2 mm2
squares. These flat wells were able to hold 2 μL of reagents,
corresponding to a height of ∼500 μm, with minimal contamination risk.
The hydrophobic ink used to pattern the surfaces allowed significantly
larger volumes of samples to be applied when compared with surfaces
patterned with nonhydrophobic inks. This reduced evaporation effects and
permitted greater pipetting accuracy, thereby improving assay
reproducibility. A competitive immunoassay was developed based on the
ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal
antibodies to 2,4-D−bovine serum albumin conjugate adsorbed onto the
glass support. The support was subsequently incubated with alkaline
phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate
bound was determined by quantitating the chemiluminescent emission
produced from the enzymatic breakdown of CSPD substrate by AP using a
cooled CCD camera. The detection limit of the single-sample microformat
assay was 2.7 × 10-11 M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10-8−4.5 × 10-11 and 4.5 × 10-7−1.66 × 10-10
M, respectively. In comparison, the detection limits of a tube-based
chemiluminescent assay using standard luminometer and of a colorimetric
ELISA were 45 × 10-11 and 9.9 × 10-8
M, respectively. The ability to scale the thick-film-based microformat
assay makes it an ideal candidate for the development of affinity arrays
and high-throughput assay formats. Prospects for further improvements
of this imaging ELISA strategy will be discussed. (Less)
Please use this url to cite or link to this publication:
author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
ELISA, 2,4-dichlorophenoxyacetic acid, Microformat imaging
in
Analytical Chemistry
volume
68
issue
19
pages
6 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:0001190926
ISSN
1520-6882
DOI
10.1021/ac960129k
language
English
LU publication?
yes
id
63c3bfcb-ca1b-47af-a789-326d6a4d44a4
date added to LUP
2024-06-17 10:10:31
date last changed
2024-06-18 04:01:25
@article{63c3bfcb-ca1b-47af-a789-326d6a4d44a4,
  abstract     = {{A flat-well microformat competitive enzyme-linked immunosorbent <br>
chemiluminescent assay for the detection of the pesticide <br>
2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film <br>
technology was used to pattern a hydrophobic layer 100 μm thick onto <br>
glass microscope slides to form an array of 2 × 2 mm<sup>2</sup> <br>
squares. These flat wells were able to hold 2 μL of reagents, <br>
corresponding to a height of ∼500 μm, with minimal contamination risk. <br>
The hydrophobic ink used to pattern the surfaces allowed significantly <br>
larger volumes of samples to be applied when compared with surfaces <br>
patterned with nonhydrophobic inks. This reduced evaporation effects and<br>
 permitted greater pipetting accuracy, thereby improving assay <br>
reproducibility. A competitive immunoassay was developed based on the <br>
ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal<br>
 antibodies to 2,4-D−bovine serum albumin conjugate adsorbed onto the <br>
glass support. The support was subsequently incubated with alkaline <br>
phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate <br>
bound was determined by quantitating the chemiluminescent emission <br>
produced from the enzymatic breakdown of CSPD substrate by AP using a <br>
cooled CCD camera. The detection limit of the single-sample microformat <br>
assay was 2.7 × 10<sup>-</sup><sup>11</sup> M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10<sup>-</sup><sup>8</sup>−4.5 × 10<sup>-</sup><sup>11</sup> and 4.5 × 10<sup>-</sup><sup>7</sup>−1.66 × 10<sup>-</sup><sup>10</sup><br>
 M, respectively. In comparison, the detection limits of a tube-based <br>
chemiluminescent assay using standard luminometer and of a colorimetric <br>
ELISA were 45 × 10<sup>-</sup><sup>11</sup> and 9.9 × 10<sup>-</sup><sup>8</sup><br>
 M, respectively. The ability to scale the thick-film-based microformat <br>
assay makes it an ideal candidate for the development of affinity arrays<br>
 and high-throughput assay formats. Prospects for further improvements <br>
of this imaging ELISA strategy will be discussed.}},
  author       = {{Dzgoev, Anatoli and Mecklenburg, Michael and Larsson, Per-Olof and Danielsson, Bengt}},
  issn         = {{1520-6882}},
  keywords     = {{ELISA; 2,4-dichlorophenoxyacetic acid; Microformat imaging}},
  language     = {{eng}},
  number       = {{19}},
  pages        = {{3364--3369}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{Analytical Chemistry}},
  title        = {{Microformat Imaging ELISA for Pesticide Determination}},
  url          = {{http://dx.doi.org/10.1021/ac960129k}},
  doi          = {{10.1021/ac960129k}},
  volume       = {{68}},
  year         = {{1996}},
}