Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals

Wong, Chin Lin, Lam, Ai Leen, Smith, Maree T. and Ghassabian, Sussan (2016) Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals. Frontiers in Pharmacology, 7 MAR: 53. doi:10.3389/fphar.2016.00053

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Author Wong, Chin Lin
Lam, Ai Leen
Smith, Maree T.
Ghassabian, Sussan
Title Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals
Journal name Frontiers in Pharmacology   Check publisher's open access policy
ISSN 1663-9812
Publication date 2016-02-26
Year available 2016
Sub-type Article (original research)
DOI 10.3389/fphar.2016.00053
Open Access Status DOI
Volume 7
Issue MAR
Start page 53
Total pages 32
Place of publication Lausanne, Switzerland
Publisher Frontiers Research Foundation
Language eng
Subject 3004 Pharmacology
2736 Pharmacology (medical)
Abstract The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Corl-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25 degrees C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Corl-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Corl-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight the difficulty in adapting in vitro methods to high-throughput format for screening the skin sensitization potential of large numbers of chemicals whilst ensuring that the data produced are both accurate and reproducible.
Keyword Allergic contact dermatitis
Cor1-C420
Cysteine
in vitro methods
Lysine
Skin Sensitization
peptide reactivity assay
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID RM20100002374
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
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Created: Mon, 14 Mar 2016, 01:12:29 EST by Professor Maree Smith on behalf of School of Pharmacy