scholarly journals A Pre-Processing Pipeline to Quantify, Visualize and Reduce Technical Variation in Protein Microarray Studies

2021 ◽  
Author(s):  
Sophie Bérubé ◽  
Tamaki Kobayashi ◽  
Amy Wesolowski ◽  
Douglas E. Norris ◽  
Ingo Ruczinski ◽  
...  
Keyword(s):  
Dna Microarrays ◽  
Protein Microarray ◽  
Protein Microarrays ◽  
Serum Samples ◽  
Fluorescent Intensity ◽  
Processing Pipeline ◽  
Infectious Disease Diagnostics ◽  
Using Data ◽  
Downstream Analysis ◽  
Microarray Studies

AbstractTechnical variation, or variation from non-biological sources, is present in most laboratory assays. Correcting for this variation enables analysts to extract a biological signal that informs questions of interest. However, each assay has different sources and levels of technical variation and the choice of correction methods can impact downstream analyses. Compared to similar assays such as DNA microarrays, relatively few methods have been developed and evaluated for protein microarrays, a versatile tool for measuring levels of various proteins in serum samples. Here, we propose a pre-processing pipeline to correct for some common sources of technical variation in protein microarrays. The pipeline builds upon an existing normalization method by using controls to reduce technical variation. We evaluate our method using data from two protein microarray studies, and by simulation. We demonstrate that pre-processing choices impact the fluorescent-intensity based ranks of proteins, which in turn, impact downstream analysis.1Impact StatementProtein microarrays are in wide use in cancer research, infectious disease diagnostics and biomarker identification. To inform research and practice in these and other fields, technical variation must be corrected using normalization and pre-processing. Current protein microarray studies use a variety of normalization methods, many of which were developed for DNA microarrays, and therefore are based on assumptions and data that are not ideal for protein microarrays. To address this issue, we develop, evaluate, and implement a pre-processing pipeline that corrects for technical variation in protein microarrays. We show that pre-processing and normalization directly impact the validity of downstream analysis, and protein-specific approaches are essential.

scholarly journals Applications of Protein Microarrays in Biomarker Discovery for Autoimmune Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Siting Li ◽  
Guang Song ◽  
Yina Bai ◽  
Ning Song ◽  
Jiuliang Zhao ◽  
...  
Keyword(s):  
Autoimmune Diseases ◽  
Biomarker Discovery ◽  
Protein Microarray ◽  
Protein Microarrays ◽  
Microarray Technology ◽  
Serum Samples ◽  
Future Studies ◽  
Key Issues

Dysregulated autoantibodies and cytokines were deemed to provide important cues for potential illnesses, such as various carcinomas and autoimmune diseases. Increasing biotechnological approaches have been applied to screen and identify the specific alterations of these biomolecules as distinctive biomarkers in diseases, especially autoimmune diseases. As a versatile and robust platform, protein microarray technology allows researchers to easily profile dysregulated autoantibodies and cytokines associated with autoimmune diseases using various biological specimens, mainly serum samples. Here, we summarize the applications of protein microarrays in biomarker discovery for autoimmune diseases. In addition, the key issues in the process of using this approach are presented for improving future studies.

scholarly journals Protein Microarrays for Personalized Medicine

2010 ◽  
Vol 56 (3) ◽  
pp. 376-387 ◽  
Author(s):  
Xiaobo Yu ◽  
Nicole Schneiderhan-Marra ◽  
Thomas O Joos
Keyword(s):  
Personalized Medicine ◽  
Protein Function ◽  
Dna Microarrays ◽  
Biomarker Discovery ◽  
Protein Microarray ◽  
Disease Diagnosis ◽  
Protein Microarrays ◽  
Nucleotide Polymorphisms ◽  
Single Experiment

Abstract Background: Over the last 10 years, DNA microarrays have achieved a robust analytical performance, enabling their use for analyzing the whole transcriptome or for screening thousands of single-nucleotide polymorphisms in a single experiment. DNA microarrays allow scientists to correlate gene expression signatures with disease progression, to screen for disease-specific mutations, and to treat patients according to their individual genetic profiles; however, the real key is proteins and their manifold functions. It is necessary to achieve a greater understanding of not only protein function and abundance but also their role in the development of diseases. Protein concentrations have been shown to reflect the physiological and pathologic state of an organ, tissue, or cells far more directly than DNA, and proteins can be profiled effectively with protein microarrays, which require only a small amount of sample material. Content: Protein microarrays have become well-established tools in basic and applied research, and the first products have already entered the in vitro diagnostics market. This review focuses on protein microarray applications for biomarker discovery and validation, disease diagnosis, and use within the area of personalized medicine. Summary: Protein microarrays have proved to be reliable research tools in screening for a multitude of parameters with only a minimal quantity of sample and have enormous potential in applications for diagnostic and personalized medicine.

scholarly journals Point-of-Care Vertical Flow Allergen Microarray Assay: Proof of Concept

2014 ◽  
Vol 60 (9) ◽  
pp. 1209-1216 ◽  
Author(s):  
Thiruppathiraja Chinnasamy ◽  
Loes I Segerink ◽  
Mats Nystrand ◽  
Jesper Gantelius ◽  
Helene Andersson Svahn
Keyword(s):  
Point Of Care ◽  
Protein Microarray ◽  
High Sensitivity ◽  
Specific Ige ◽  
Protein Microarrays ◽  
Vertical Flow ◽  
Proof Of Concept ◽  
Serum Samples ◽  
Clinical Model ◽  
Assay Time

Abstract BACKGROUND Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of <10 min before imaging and data analysis. METHOD Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of ≤10 min. Microarray images were captured by a consumer-grade flatbed scanner. RESULTS A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was <14%. The observed concordance with a clinical assay, ImmunoCAP, was R2 = 0.89 (n = 31). CONCLUSIONS In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.

Method to minimize ozone effect on Cy5 fluorescent intensity in DNA microarrays

2017 ◽  
Vol 538 ◽  
pp. 1-4
Author(s):  
Youngjun Kim ◽  
Hyun Hee Seo ◽  
Mi Seon Jeong ◽  
Ki Heon Lee ◽  
In Ho Lee ◽  
...  
Keyword(s):  
Dna Microarrays ◽  
Fluorescent Intensity

Protein Microarrays Printed from DNA Microarrays

2010 ◽  
pp. 95-106 ◽  
Author(s):  
Oda Stoevesandt ◽  
Mingyue He ◽  
Michael J. Taussig
Keyword(s):  
Dna Microarrays ◽  
Protein Microarrays

scholarly journals Serological Levels of Anti-clathrin Antibodies Are Decreased in Patients With Pseudoexfoliation Glaucoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Vanessa M. Beutgen ◽  
Norbert Pfeiffer ◽  
Franz H. Grus
Keyword(s):  
Open Angle Glaucoma ◽  
Protein Microarray ◽  
Enrichment Analysis ◽  
Normal Tension Glaucoma ◽  
Control Group ◽  
Open Angle ◽  
Pseudoexfoliation Glaucoma ◽  
Serum Samples ◽  
Protein Protein Interaction ◽  
Circulating Autoantibodies

Evidence for immunologic contribution to glaucoma pathophysiology is steadily increasing in ophthalmic research. Particularly, an altered abundance of circulating autoantibodies to ocular antigens is frequently observed. Here, we report an analysis of autoantibody abundancies to selected antigens in sera of open-angle glaucoma patients, subdivided into normal-tension glaucoma (N = 31), primary open-angle glaucoma (N = 43) and pseudoexfoliation glaucoma (N = 45), vs. a non-glaucomatous control group (N = 46). Serum samples were analyzed by protein microarray, including 38 antigens. Differences in antibody levels were assessed by ANOVA. Five serological antibodies showed significantly altered levels among the four groups (P < 0.05), which can be used to cluster the subjects in groups consisting mainly of PEXG or POAG/NTG samples. Among the altered autoantibodies, anti-Clathrin antibodies were identified as most important subgroup predictors, enhancing prospective glaucoma subtype prediction. As a second aim, we wanted to gain further insights into the characteristics of previously identified glaucoma-related antigens and their role in glaucoma pathogenesis. To this end, we used the bioinformatics toolset of Metascape to construct protein-protein interaction networks and GO enrichment analysis. Glaucoma-related antigens were significantly enriched in 13 biological processes, including mRNA metabolism, protein folding, blood coagulation and apoptosis, proposing a link of glaucoma-associated pathways to changes in the autoantibody repertoire. In conclusion, our study provides new aspects of the involvement of natural autoimmunity in glaucoma pathomechanisms and promotes advanced opportunities toward new diagnostic approaches.

scholarly journals Instrument-Free Protein Microarray Fabrication for Accurate Affinity Measurements

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 158
Author(s):  
Iris Celebi ◽  
Matthew T. Geib ◽  
Elisa Chiodi ◽  
Nese Lortlar Ünlü ◽  
Fulya Ekiz Kanik ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Low Cost ◽  
Protein Microarray ◽  
Active Surface ◽  
Protein Microarrays ◽  
Free Protein ◽  
Microarray Fabrication ◽  
Antigen Antibody ◽  
Imaging Sensor ◽  
Protein Assays

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have a higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in a 24-multiplexed chip format with less than 5% measurement error.

A critical comparison of protein microarray fabrication technologies

The Analyst ◽  
2014 ◽  
Vol 139 (6) ◽  
pp. 1303-1326 ◽  
Author(s):  
Valentin Romanov ◽  
S. Nikki Davidoff ◽  
Adam R. Miles ◽  
David W. Grainger ◽  
Bruce K. Gale ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Microarray ◽  
Protein Microarrays ◽  
Antibody Affinity ◽  
High Throughput Analysis ◽  
Critical Comparison ◽  
Binding Partners ◽  
Fundamental Information ◽  
Microarray Fabrication ◽  
Analytical Tools

Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis.

Intra- and interlaboratory sources of imprecision in drug measurements by different techniques

1993 ◽  
Vol 39 (5) ◽  
pp. 851-855 ◽  
Author(s):  
L M Tsanaclis ◽  
J F Wilson
Keyword(s):  
External Quality Assessment ◽  
Serum Samples ◽  
External Quality Assessment Scheme ◽  
Drug Concentrations ◽  
Bartlett’S Test ◽  
Homogeneity Of Variance ◽  
Sources Of Variation ◽  
Laboratory Procedures ◽  
Assay Precision ◽  
Using Data

Abstract We compared the intra- and interlaboratory precision of seven techniques used to measure eight antiepileptic drugs, digoxin, and theophylline by using data from the international Healthcontrol external quality-assessment scheme. Scheme participants were supplied blind with 6 or 12 sets of duplicate lyophilized serum samples. Each set contained different drug concentrations, and duplicates were analyzed separately, 1 to 6 months apart. The intra- and interlaboratory components of assay variance were isolated and compared by Bartlett's test for homogeneity of variance. Fluorescence polarization immunoassay (Abbott) showed the best overall intra- and interlaboratory performance for a range of analytes. The largest intralaboratory errors were produced by techniques using the Syva EMIT assays. Our analysis of the data shows that for most analyte/technique combinations, intralaboratory sources of variation were more important than interlaboratory sources. Gains in assay precision will therefore result from attention to internal laboratory procedures.

scholarly journals Navigating Public Microarray Databases

2004 ◽  
Vol 5 (6-7) ◽  
pp. 471-479 ◽  
Author(s):  
Christopher J. Penkett ◽  
Jürg Bähler
Keyword(s):  
Data Sources ◽  
European Bioinformatics Institute ◽  
Published Data ◽  
Expression Data ◽  
The Public ◽  
Genome Wide ◽  
Public Repositories ◽  
Set Up ◽  
Downstream Analysis ◽  
Microarray Studies

With the ever-escalating amount of data being produced by genome-wide microarray studies, it is of increasing importance that these data are captured in public databases so that researchers can use this information to complement and enhance their own studies. Many groups have set up databases of expression data, ranging from large repositories, which are designed to comprehensively capture all published data, through to more specialized databases. The public repositories, such as ArrayExpress at the European Bioinformatics Institute contain complete datasets in raw format in addition to processed data, whilst the specialist databases tend to provide downstream analysis of normalized data from more focused studies and data sources. Here we provide a guide to the use of these public microarray resources.

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