Affinity-capture reagents for protein arrays

Elia, Giuliano; Silacci, Michela; Scheurer, Simone; Scheuermann, Jörg; Neri, Dario

doi:10.1016/s1471-1931(02)00201-x

Cited by 35 publications

(15 citation statements)

References 26 publications

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“…Indeed, covalently attaching proteins to a surface often results in a loss of biological activity (3,4,22,29). To determine if trypsin immobilization to the methacrylate polymer also retains protein activity, trypsin was immobilized in methacrylate element arrays and incubated with 10 M apomyoglobin overnight at 37C.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Protein Interaction and Function with a 3-Dimensional MALDI-MS Protein Array

et al. 2005

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Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.

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Section: Resultsmentioning

confidence: 99%

Analysis of Protein Interaction and Function with a 3-Dimensional MALDI-MS Protein Array

et al. 2005

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“…New immuno-depletion strategies efficiently remove as many as 20 of the high-abundance constituents [11]. Techniques for immunoextraction and concentration of targeted biomarker fragments may be more reliable [12][13][14][15][16][17]. However, the degree to which relevant low-abundance proteins are lost during processing to remove highabundance proteins is unclear and may be highly variable.…”

Section: Challenges Inherent To Plasma Proteomicsmentioning

confidence: 99%

Advantages of Multiplex Proteomics in Clinical Immunology

Lea¹,

Keystone

Mudumba³

et al. 2009

Clinic Rev Allerg Immunol

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Clinical multiplex diagnostic proteomics is the application of proteomic technologies to improve a patient's clinical outcomes. The future holds impact potential for testing prognosis, diagnosis, and drug therapy, while monitoring efficacious treatment with qualitative and quantitative data. Multiplex clinical diagnostic use of novel biomarkers in body fluids to confirm presence and severity of clinical disease states, holds great promise for clinical use. Challenges for diagnostic clinics include awareness of proteome complexity in clinical samples, the effects of high-abundance proteins, such as albumin, that could mask detection of other and low abundance disease proteins or biomarkers. Standardized approaches to sample collection and preparation, new analytical techniques and novel algorithms for bio-statistical analysis will facilitate release of the great potential of clinical multiplex diagnostic proteomics. A sensitive RA assay has been developed for the simultaneous measurement of the three rheumatoid factors (RFs), RF-IgA, IgG, and IgM, with the option to simultaneously measure anti-cyclic citrullinated peptide (anti-CCP) IgG antibodies using IgXPLEX™: technology. Testing 10-μL serum samples, SQI's multiplex microarray rheumatoid arthritis assay provides both positive/negative as well as qualitative/semi-quantitative results for anti-CCP IgG, RF-IgA, IgG, and IgM in each sample well on a 96-well microtiter-formatted microarray plate. Signal detection uses sensitive fluorescent-tagged markers captured onto planar microarray spots and read in a microarray scanner. Each result is verified with confidence confirmation technology and validating quality controls in every sample well. For an 80-RA positive patient cohort, the 4-PLEX profile sensitivity was determined at 82.5%. The specificity for the 44 RA healthy control cohort was determined at 97.7%. The multiplex data also demonstrated that a patients' severity of disease profile, mild to severe, correlates the status of RA biomarkers to disease status.

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“…Secondly, this also enables one to operate with high selectivity (as the selectivity is based upon binding constants). Hence, it is not surprising that antibodies have been the most frequently used affinity ligands in protein arrays so far [94,95]. One can generate antibodies which are highly specific without worrying about the low dissociation constants of the resulting affinity complexes.…”

Section: Challenges In Design Of Protein Arraysmentioning

confidence: 99%