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Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-1855-703X
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8141-8449
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2009 (English)In: Clinical and Laboratory 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. Vol. 16, no 11, 1665-1674 p.
Keyword [en]
contagious bovine pleuropneumonia, lipoprotein lppq, genetic-characterization, sc, diagnosis, elisa, cbpp, capricolum, tanzania, cluster
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11105DOI: 10.1128/CVI.00223-09ISI: 000271373700019Scopus ID: 2-s2.0-70350702934OAI: oai:DiVA.org:kth-11105DiVA: diva2:235811
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: 2013-02-08Bibliographically approved
In thesis
1. Protein based approaches to understand and prevent contagious bovine pleuropneumonia
Open this publication in new window or tab >>Protein based approaches to understand and prevent contagious bovine pleuropneumonia
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. x, 77 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:17
Keyword
CBPP, Mycoplasma mycoides subsp. mycoides small colony type, humoral immune responses, recombinant surface proteins, ELISA, subunit vaccine, suspension bead array
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11108 (URN)978-91-7415-417-7 (ISBN)
Public defence
2009-10-16, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100719Available from: 2009-09-25 Created: 2009-09-18 Last updated: 2010-07-19Bibliographically approved
2. Bead based protein profiling in blood
Open this publication in new window or tab >>Bead based protein profiling in blood
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
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:nbn:se:kth:diva-117960 (URN)978-91-7501-629-0 (ISBN)
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

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