Development of miniaturized immunoassay: Influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot

Khoury, Graziella El; Laurenceau, Emmanuelle; Chevolot, Yann; Mérieux, Yves; Desbos, Agnès; Fabien, Nicole; Rigal, Dominique; Souteyrand, Éliane; Cloarec, Jean-Pierre

doi:10.1016/j.ab.2010.01.013

Cited by 17 publications

(12 citation statements)

References 27 publications

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“…Alternatively, microstructured glass slides were silanized with tert-butyl-11-(dimethylamino) silylundecanoate (TDSUM) and carboxylic acids were deprotected as described previously 9,[25][26][27] .…”

Section: Surface Functionalization Of Microstructured Glass Slidementioning

confidence: 99%

Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

et al. 2013

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Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.

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Section: Surface Functionalization Of Microstructured Glass Slidementioning

confidence: 99%

Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

et al. 2013

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“…For example, improvements have been reported concerning the substratum and data capture. Functionalized glass (36,37), hydrogel (38,39), PVDF (40,41), macroporous silicon (42), nitrocellulose polymers (43,44) (Grace Bio-Labs; Maine Manufacturing; Sartorius), and planar wave guide surfaces (ZEPTOSENS) (45) have all been successfully implemented to improve sensitivity, spot morphology, precision, and accuracy. Further marked improvements have been made in informatics approaches to deal with sample handling, regional staining correction, quality control, and the identification of high-quality samples and reagents.…”

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confidence: 99%

Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

Akbani

Becker

Carragher

et al. 2014

Molecular & Cellular Proteomics

152

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Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories.Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following:• preservation and optimization of pre-analytical sample quality,• application of validated high-affinity and specific antibody (or other protein affinity) detection reagents,• dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and• quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments.In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. Molecular & Cellular Proteomics 13: 10.1074/mcp.O113.034918, 1625-1643, 2014.Reverse phase protein array (RPPA) 1 technology represents a highly efficient and cost-effective descendent of miniaturized immunoassays that use a sandwich format for antigen capture (1, 2). Immunoassay arrays were generally sandwichstyle assays in which a series of antibodies immobilized on the solid phase were used to capture the analyte of interest ("antibody capture"), with a second antibody, directed against a different epitope on the same protein, used as a detection molecule. In contrast, in "reverse phase" the analytes (antigens) are immobilized on the solid phase (usually nitrocellulose) and subsequently probed with an antibody or other affini...

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“…Furthermore, COOH, NHS and APDMES surfaces are composed of silane monolayer whereas chitosan, CMD and MAMVE surfaces were grafted with hydrophilic polymers. Indeed, polymers and multilayer coated surfaces were shown to better maintain the native conformation of proteins and their biological activity [34][35][36][37]. In addition to the immobilization strategy, the physicochemical characteristics of captured proteins (size, structure, charge) greatly influence immobilization efficiency and biological activity [28,29,38].…”

Section: Optimization Of Tumor Antigens Microarray Conditionsmentioning

confidence: 99%

Anti-heat shock protein autoantibody profiling in breast cancer using customized protein microarray

et al. 2015

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Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%.

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Development of miniaturized immunoassay: Influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot

Cited by 17 publications

References 27 publications

Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

Anti-heat shock protein autoantibody profiling in breast cancer using customized protein microarray

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