Shera Lilyanna1, Enoch Ming Wei Ng2, Shiho Moriguchi3, Siew Pang Chan1, Ryohei Kokawa4, So Hung Huynh2, P C Jenny Chong1, Yan Xia Ng1, A Mark Richards1,5, Tuck Wah Ng2, Oi Wah Liew1. 1. Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore. 2. Laboratory for Optics & Applied Mechanics, Department of Mechanical & Aerospace Engineering, Monash University, Clayton, Australia. 3. Shimadzu Techno-Research Inc., Kyoto, Japan. 4. Global Application Development Center, Shimadzu Corporation, Japan. 5. Christchurch Heart Institute, Department of Medicine, University of Otago, Christchurch, New Zealand.
Abstract
BACKGROUND: Microplate-based immunoassays are widely used in clinical and research settings to measure a broad range of biomarkers present in complex matrices. Assay variability within and between microplates can give rise to false-negative and false-positive results leading to incorrect conclusions. To date, the contribution of microplates to this variability remains poorly characterized and described. This study provides new insights into variability in immunoassays attributable to surface characteristics of commercial microplates. METHODS: Well-to-well assay variation in γ-treated and nontreated 96-well opaque microplates suitable for chemiluminescence assays was determined by use of a validated sandwich ELISA. Microplate surface characteristics were assessed by sessile drop contact angle measurements, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic force microscopy. RESULTS: All microplate types tested exhibited vendor-specific assay response profiles; and "rogue" plates with very high intraassay variation and deviant mean assay responses were found. Within-plate, location-dependent bias in assay responses and variability in well contact angle were also observed. We demonstrate substantial differences in well-surface properties with putative effects on protein-coating reproducibility and hence consistency in immunoassay responses. A surface "cleaning" effect on manufacturing residues was attributed to γ-irradiation, and treated microplates manifest increased polar functionalities, surface roughness, and assay responses. CONCLUSIONS: Our data suggest that tighter control of variability in surface roughness, wettability, chemistry, and level of residual contaminants during microplate preparation is warranted to improve consistency of ELISA assay read out.
BACKGROUND: Microplate-based immunoassays are widely used in clinical and research settings to measure a broad range of biomarkers present in complex matrices. Assay variability within and between microplates can give rise to false-negative and false-positive results leading to incorrect conclusions. To date, the contribution of microplates to this variability remains poorly characterized and described. This study provides new insights into variability in immunoassays attributable to surface characteristics of commercial microplates. METHODS: Well-to-well assay variation in γ-treated and nontreated 96-well opaque microplates suitable for chemiluminescence assays was determined by use of a validated sandwich ELISA. Microplate surface characteristics were assessed by sessile drop contact angle measurements, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic force microscopy. RESULTS: All microplate types tested exhibited vendor-specific assay response profiles; and "rogue" plates with very high intraassay variation and deviant mean assay responses were found. Within-plate, location-dependent bias in assay responses and variability in well contact angle were also observed. We demonstrate substantial differences in well-surface properties with putative effects on protein-coating reproducibility and hence consistency in immunoassay responses. A surface "cleaning" effect on manufacturing residues was attributed to γ-irradiation, and treated microplates manifest increased polar functionalities, surface roughness, and assay responses. CONCLUSIONS: Our data suggest that tighter control of variability in surface roughness, wettability, chemistry, and level of residual contaminants during microplate preparation is warranted to improve consistency of ELISA assay read out.
Authors: Tamara van Gorkom; Gijs H J van Arkel; Willem Voet; Steven F T Thijsen; Kristin Kremer Journal: J Clin Microbiol Date: 2021-07-19 Impact factor: 5.948
Authors: Oi Wah Liew; Samantha S M Ling; Shera Lilyanna; Yue Zhou; Peipei Wang; Jenny P C Chong; Yan Xia Ng; Angeline E S Lim; Eliot R Y Leong; Qifeng Lin; Teck Kwang Lim; Qingsong Lin; Enoch M W Ng; Tuck Wah Ng; A Mark Richards Journal: Commun Biol Date: 2021-04-06