Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions

By B. Haab, Maitreya J. Dunham, Patrick O. Brown. Genome Biology, 2001

880 citations recorded by the source corpus.

BackgroundWe have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes.ResultsSpecific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 μg/ml and 1.6 μg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.ConclusionsThese results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.

Subfields: Biomedical Engineering, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/501c9e225aa6ad19b63e37ba68e41b7ab4fcb70a

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Citations in the corpus

A new method for label-free imaging of biomolecular interactions (2003, background): “Likewise, arrays of hybridized protein probes are finding applications in protein pathway analysis[3], and protein expression diagnostic tests[4].”
Generation and analyses of human synthetic antibody libraries and their application for protein microarrays. (2016, background): “As for example, early work showed that 20% or even less of the monoclonal and polyclonal antibodies tested did not work well in arraybased applications (Haab et al., 2001; Borrebaeck and Wingren, 2007).”
Biosensors for Protein Detection: A Review (2005, methodology): “based on immunoreactions, with optical detection (Haab et al. 2001), electrochemical detection (Kojima et al.”
Cell and organ printing 1: protein and cell printers. (2003, methodology): “…making protein or DNA chips includes photolithography, which is mainly used for synthesis of oligo DNA chips (Southern, 1996; Gao et al., 2001), and pin arrayers (Shalon et al., 1996), which have been used as a platform for both cDNA and protein arrays (Wilson and Nock, 2002; Haab et al., 2001).”
Autoantigen microarrays for multiplex characterization of autoantibody responses (2002, background): “Haab et al.(8) developed spotted-array-based quantification of 115 antigen–antibody pairs.”
BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data (2002, methodology): “The technology has been utilized in various applications such as the profiling of mRNA [2] and protein levels [3], elucidating protein-DNA interactions [4], assessment of DNA copy number [5], and detection of methylated sequences [6], and today is accessible to even relatively small laboratories.”
Proteomics: the first decade and beyond (2003, background): “It therefore seemed obvious to apply similar principles to proteomic analyses—in particular to protein expression profiling—by generating ordered arrays of specific protein-binding module”
Protein microarrays and proteomics (2002, background): “In one of the earliest and most extensive attempts to examine the feasibility of a two-color labeling approach, Haab et al”
Designing materials for biology and medicine (2004, background): “Brown and co-workers examined the simple approach of printing proteins (either antigens or antibodies) on poly( l -lysine)-coated glass microscope slide”