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Bead based protein profiling in blood
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab. (Biobank Profiling)ORCID iD: 0000-0002-1855-703X
2013 (Swedish)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles.

A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. , 116 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:4
Keyword [en]
Affinity proteomics, protein array, suspension bead array, antigen, antibody, biomarker discovery, serology, selectivity, sensitivity, serum, plasma
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-117960ISBN: 978-91-7501-629-0 (print)OAI: oai:DiVA.org:kth-117960DiVA: diva2:604032
Public defence
2013-03-01, Gardaulan, Smittskyddsinstitutet, Nobels väg 18, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20130208

Available from: 2013-02-08 Created: 2013-02-07 Last updated: 2013-02-08Bibliographically approved
List of papers
1. Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay
Open this publication in new window or tab >>Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay
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2009 (English)In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 16, no 11, 1665-1674 p.Article in journal (Refereed) Published
Abstract [en]

A recombinant antigen cocktail ELISA for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one third of the surface proteome of the infectious agent Mycoplasma mycoides subsp. mycoides SC (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for their humoral immune response towards 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with p-values less than 10-6. Only minor cross reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a five fold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origin. No false positives and only two false negatives were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offer a powerful combination to drive and further improve serological assays towards reliable, simple and cost-effective diagnosis of CBPP.

Place, publisher, year, edition, pages
Washington D.C.: American Society for Microbiology, 2009
Keyword
contagious bovine pleuropneumonia, lipoprotein lppq, genetic-characterization, sc, diagnosis, elisa, cbpp, capricolum, tanzania, cluster
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11105 (URN)10.1128/CVI.00223-09 (DOI)000271373700019 ()2-s2.0-70350702934 (Scopus ID)
Note
QC 20100719. Updated from in press to published. Previous title "Multiplex screening of surface proteins from Mycoplasma mycoides subsp. mycoides SC for an antigen cocktail ELISA"Available from: 2009-09-18 Created: 2009-09-18 Last updated: 2017-12-13Bibliographically approved
2. Plasma Profiling Reveals Human Fibulin-1 as Candidate Marker for Renal Impairment
Open this publication in new window or tab >>Plasma Profiling Reveals Human Fibulin-1 as Candidate Marker for Renal Impairment
Show others...
2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 11, 4925-4934 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.

Keyword
affinity proteomics, plasma profiling, antibody microarray, kidney disorders, biomarker, fibulin-1, glomerulonephritis
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-51288 (URN)10.1021/pr200286c (DOI)000296414700004 ()2-s2.0-80655139676 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20111212Available from: 2011-12-12 Created: 2011-12-12 Last updated: 2017-12-08Bibliographically approved
3. Classification of protein profiles from antibody microarrays using heat and detergent treatment.
Open this publication in new window or tab >>Classification of protein profiles from antibody microarrays using heat and detergent treatment.
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2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 564-570 p.Article in journal (Refereed) Published
Abstract [en]

Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-52450 (URN)10.1016/j.nbt.2011.10.005 (DOI)000305606500008 ()22023822 (PubMedID)2-s2.0-84862011672 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20111216Available from: 2011-12-16 Created: 2011-12-16 Last updated: 2017-12-08Bibliographically approved
4. Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysis
Open this publication in new window or tab >>Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysis
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The increasing availability of antibodies towards human proteins drives the generation of new and exploratory data, which can be generated by profiling e.g. plasma samples using multiplexed arrays. However, antibody assays can lead to erroneous results due to cross-­‐reactivity to off-­‐targets. Here, we describe an approach in which an antibody-­‐based suspension bead array is combined with subsequent validation of on-­‐ target binding using a coupled affinity purification assay. Based on differential heat treatment of samples, antibody performance was investigated and the results for antibodies directed towards several complement factors provide insights on the detection of proteins in sera from patients with complement deficiency. In conclusion, the combined parallel flow and blot based immunocapture analysis serves an important, first line tool for resolving differently detected serum or plasma protein profiles proposed by antibody arrays. 

Keyword
Suspension bead array, protein profiling, antibody, immunocapture, Western blot
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-117959 (URN)
Note

QS 2013

Available from: 2013-02-07 Created: 2013-02-07 Last updated: 2013-02-08Bibliographically approved

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