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On Generation and Applications of High-Density Protein Microarrays
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0003-1363-5796
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affinity proteomics has experienced rapid development over the last two decades and one of the most promising platforms to emerge are the protein microarrays. The combination of affinity reagents and miniaturisation enables assays for simultaneous high throughput and sensitive protein analysis. Due to the combination of these desrable properties, a multitude of protein array platforms for rapid and efficient study of proteomes and protein interactions are in use today. Although the protein microarray field has more than two decades of history to look back on the development of new protein microarray platforms continues to this day and beyond.

In the paper I in this thesis, a microarray of eluates from dried blood spot samples collected from neonates were designed and utilised for detection of complement factor 3 (C3) deficiency. The data acquired from the microarrays platform were compared to C3 levels obtained through enzyme-linked immunosorbant assay (ELISA), and the microarray assay were found to separate the C3 deficient samples from the controls. The conclusion of this investigation was that the microarray platform would be suitable for high-throughput screening of C3 deficiency in neonates. Paper II outlines the work in developing a multiplex platform for validation of affinity reagents. A set of 398 affinity binders, originating from five research groups, were profiled against 432 antigens and representing both polyclonal rabbit antibodies, monoclonal mouse antibodies, and recombinant single-chain variable fragments. Approximately 50% of the binders were found to preferably recognise their intended target while 10% of the binders did not generate any, or low, signals with their respective targets. For paper III, a reverse phase array (RPPA) platform using fluorescence-based detection of IgA deficiency in over 2.000 samples where validated on a label-free detection system and ELISA. The data from the label-free platform and the RPPA were found agree well with each other while data from ELISA did with neither of them. It was found that the label-free platform proved to be well-suited for detection of IgA in serum. Paper IV describes one of the world’s largest protein microarrays containing 21.120 recombinant protein fragments. We describe some of the possible applications of these large-scale arrays, such as binding profiles for the validation of antibodies with 11.520 and 21.120 recombinant proteins, as well as screening for autoimmunity in human serum samples.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , ix, 63 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:12
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-168165ISBN: 978-91-7595-579-7 (print)OAI: oai:DiVA.org:kth-168165DiVA: diva2:814541
Public defence
2015-06-12, Inghe-salen, Tomtebodavägen 18a, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20150528

Available from: 2015-05-28 Created: 2015-05-27 Last updated: 2016-01-28Bibliographically approved
List of papers
1. Screening for C3 deficiency in newborns using microarrays.
Open this publication in new window or tab >>Screening for C3 deficiency in newborns using microarrays.
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2009 (English)In: PloS one, ISSN 1932-6203, Vol. 4, no 4, e5321- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Dried blood spot samples (DBSS) from newborns are widely used in neonatal screening for selected metabolic diseases and diagnostic possibilities for additional disorders are continuously being evaluated. Primary immunodeficiency disorders comprise a group of more than one hundred diseases, several of which are fatal early in life. Yet, a majority of the patients are not diagnosed due to lack of high-throughput screening methods.

METHODOLOGY/PRINCIPAL FINDINGS: We have previously developed a system using reverse phase protein microarrays for analysis of IgA levels in serum samples. In this study, we extended the applicability of the method to include determination of complement component C3 levels in eluates from DBSS collected at birth. Normal levels of C3 were readily detected in 269 DBSS from healthy newborns, while no C3 was detected in sera and DBSS from C3 deficient patients.

CONCLUSIONS/SIGNIFICANCE: The findings suggest that patients with deficiencies of specific serum proteins can be identified by analysis of DBSS using reverse phase protein microarrays.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-83827 (URN)10.1371/journal.pone.0005321 (DOI)000265514400009 ()19390687 (PubMedID)
Note

QC 20120213

Available from: 2012-02-13 Created: 2012-02-13 Last updated: 2015-05-28Bibliographically approved
2. Validation of affinity reagents using antigen microarrays
Open this publication in new window or tab >>Validation of affinity reagents using antigen microarrays
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2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 555-563 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

Keyword
protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-48322 (URN)10.1016/j.nbt.2011.11.009 (DOI)000305606500007 ()2-s2.0-84862014285 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Available from: 2011-11-17 Created: 2011-11-17 Last updated: 2017-12-08Bibliographically approved
3. Biosensor based protein profiling on reverse phase serum microarray
Open this publication in new window or tab >>Biosensor based protein profiling on reverse phase serum microarray
2012 (English)In: Journal of Proteomics & Bioinformatics, ISSN 0974-276X, E-ISSN 0974-276X, Vol. 5, no 8, 185-189 p.Article in journal (Refereed) Published
Abstract [en]

The reverse phase serum microarray format enables multi-parallel and simultaneous analysis of literally thousands of samples, a feature which is of uttermost importance for protein profiling of clinical samples. We have here screened 2400 serum samples for their potential IgA deficiency by using a fluorescence based reverse phase serum microarray platform and a biosensor based label-free microarray platform for verification and also compared our microarray-results to clinical routine ELISA. We have been able to identify possible IgA-deficiencies and to show the suitability of our microarray-platforms for large-scale screening of clinical serum samples. The two microarray methods show reproducibility and correlation towards each other and low variation between replicates within each platform. Both of the microarray platforms show less agreement towards ELISA. The fluorescence based microarray method has been shown to be applicable for large-scale screening of clinically important serum samples for detection of possibly IgA-deficient patients. Furthermore, it was found that the microarray based biosensor method could be used for determining the relative differences in concentration of IgA between the samples.

Keyword
Biosensor, Label-free, Protein profiling and screening, Reverse phase serum microarray, SPR
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-103635 (URN)10.4172/jpb.1000233 (DOI)2-s2.0-84866123312 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121017

Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2017-12-07Bibliographically approved
4. Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling
Open this publication in new window or tab >>Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-168187 (URN)
Note

QS 2015

Available from: 2015-05-27 Created: 2015-05-27 Last updated: 2016-11-29Bibliographically approved

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