Microplate washing: process description and improvements

Beumer, Tom; Stoffelen, Eddy; Smits, Joost; Carpay, Wim

doi:10.1016/0022-1759(92)90215-f

Cited by 5 publications

(9 citation statements)

References 14 publications

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“…At high frequencies, M becomes independent of the filling volume. This is in accordance with earlier practical and theoretical findings on the motion pattern of fluids in this type of shakers, 9,15 where oscillation amplitudes and velocities of the liquid are limited by the height of the well. Theories on turbulence suggest that good mixing requires turbulent liquid motion, 19,20 which in turn matches with thinnest laminar flow boundary layers.…”

Section: Resultssupporting

confidence: 93%

“…Immunoassays. Since enzyme conjugate concentrations in sensitive enzyme immunoassays usually are high, 9 no significant diffusion and mixing effects were to be expected during conjugate incubation. 11 We therefore only studied the effects of convection during sample incubation.…”

Section: Methodsmentioning

confidence: 99%

“…A second, equally important assay property is the interassay variation. All assay processing steps like incubation times, sample volume control, and washing out of free conjugate 9 have generally been accepted as sources of interassay variation. Yet, the effect of incubation technology never got the attention it deserved.…”

mentioning

confidence: 99%

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Convection during Incubation of Microplate Solid Phase Immunoassay: Effects on Assay Response and Variation

Beumer¹,

Haarbosch²,

Carpay³

1996

Anal. Chem.

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Section: Resultssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

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Convection during Incubation of Microplate Solid Phase Immunoassay: Effects on Assay Response and Variation

Beumer¹,

Haarbosch²,

Carpay³

1996

Anal. Chem.

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“…Copeland (1998) emphasized that flaws in the performance of mechanical washers are a common source of elevated background absorbance values, and that even where a plate washer can aspirate the contents of wells, it is better to empty them by hand. Beumer et al (1992) also noted many shortcomings in mechanical plate washers and improvements that might be made to them. The present data suggest that a cycle of three machine washes allows a significant fraction of nonspecific reactants to remain in the wells and that two further machine washes give little improvement.…”

Section: Discussionmentioning

confidence: 99%

Improved detection of citrus psorosis virus using polyclonal and monoclonal antibodies

Alioto¹,

Gangemi

Deaglio

et al. 1999

Plant Pathology

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Citrus psorosis is a serious and widespread disease associated with citrus psorosis virus (CPsV), a novel filamentous negative-stranded virus in the genus Ophiovirus. Laborious and costly indexing on test plants has been the only routine diagnostic method available, but recently an antiserum usable in double antibody sandwich (DAS) ELISA has been prepared. Here, major improvements to the DAS-ELISA protocol, a new purification method, and production of two monoclonal antibodies (mabs) to CPsV, an IgG and an IgM are reported. A highly sensitive triple antibody sandwich (TAS) ELISA making use of the mabs is described. In glasshouse citrus the homologous virus was still detectable at a tissue dilution of 1/6250 in DAS and at 1/31250 in TAS-ELISA. Both the DAS and IgG mab-TAS formats detected all CPsV isolates so far tested (from Argentina, Italy, Lebanon, Spain and the USA). A few isolates were not detected by the IgM mab.

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“…A major difficulty with immunoassays is assay variation attributed to both microplate-and nonmicroplate-associated factors, and compounded by a complicity of these factors leading to exacerbation of assay variability. Operator competency; microplate, calibrator, and reagent lot differences; microplate edge effects arising from temperature and evaporation rate differentials; and convective forces during assay incubation and washing steps have been reported to contribute to variations in well-to-well and plateto-plate signal read out (1)(2)(3)(4)(5)(6)(7)(8). Microplate variability with row and/or column effects within the microplate as well as systematic differences between microplates has been observed (7,8).…”

mentioning

confidence: 99%

Variability in Microplate Surface Properties and Its Impact on ELISA

Lilyanna

Moriguchi

et al. 2018

The Journal of Applied Laboratory Medicine

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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 γirradiation, 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. IMPACT STATEMENT In clinical and research settings, microplate-based immunoassays are subject to assay variability within and between microplates that can give rise to false-negative and false-positive results leading to incorrect conclusions. This article clarifies the contribution of microplates to assay variability, which is poorly understood to date. Detailed physicochemical analyses of microplate well surfaces provided new insights into immunoassay variability attributable to microplate surface properties and preparation processes. Our work prompts further investigations to elucidate surface parameters that influence antibody adsorption and biofunctionality, highlighting manufacturing issues that require further review to overcome problems with microplate variability and assay performance.

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Microplate washing: process description and improvements

Cited by 5 publications

References 14 publications

Convection during Incubation of Microplate Solid Phase Immunoassay: Effects on Assay Response and Variation

Convection during Incubation of Microplate Solid Phase Immunoassay: Effects on Assay Response and Variation

Improved detection of citrus psorosis virus using polyclonal and monoclonal antibodies

Variability in Microplate Surface Properties and Its Impact on ELISA

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