Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals
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 an...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-03-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fphar.2016.00053/full |
| _version_ | 1818250272901693440 |
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| author | Chin Lin eWong Chin Lin eWong Ai Leen eLam Maree Therese Smith Maree Therese Smith Sussan eGhassabian |
| author_facet | Chin Lin eWong Chin Lin eWong Ai Leen eLam Maree Therese Smith Maree Therese Smith Sussan eGhassabian |
| author_sort | Chin Lin eWong |
| collection | DOAJ |
| description | 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 Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA) and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-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 Cor1-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. |
| first_indexed | 2024-12-12T15:49:46Z |
| format | Article |
| id | doaj.art-49a2ce7d2d204ae9a82d7881aec2ebdc |
| institution | Directory Open Access Journal |
| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-12-12T15:49:46Z |
| publishDate | 2016-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Pharmacology |
| spelling | doaj.art-49a2ce7d2d204ae9a82d7881aec2ebdc2022-12-22T00:19:40ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122016-03-01710.3389/fphar.2016.00053169737Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicalsChin Lin eWong0Chin Lin eWong1Ai Leen eLam2Maree Therese Smith3Maree Therese Smith4Sussan eGhassabian5The University of QueenslandThe University of QueenslandThe University of QueenslandThe University of QueenslandThe University of QueenslandThe University of QueenslandThe 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 Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA) and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-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 Cor1-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.http://journal.frontiersin.org/Journal/10.3389/fphar.2016.00053/fullCysteineLysineIn vitro methodsAllergic contact dermatitisSkin sensitizationCor1-C420 |
| spellingShingle | Chin Lin eWong Chin Lin eWong Ai Leen eLam Maree Therese Smith Maree Therese Smith Sussan eGhassabian Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals Frontiers in Pharmacology Cysteine Lysine In vitro methods Allergic contact dermatitis Skin sensitization Cor1-C420 |
| title | Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals |
| title_full | Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals |
| title_fullStr | Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals |
| title_full_unstemmed | Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals |
| title_short | Evaluation of a high-throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals |
| title_sort | evaluation of a high throughput peptide reactivity format assay for assessment of the skin sensitization potential of chemicals |
| topic | Cysteine Lysine In vitro methods Allergic contact dermatitis Skin sensitization Cor1-C420 |
| url | http://journal.frontiersin.org/Journal/10.3389/fphar.2016.00053/full |
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