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Detection of individual microbial pathogens by proximity ligation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2006 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 52, no 6, 1152-1160 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity.

METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents.

RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium.

CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

Place, publisher, year, edition, pages
2006. Vol. 52, no 6, 1152-1160 p.
Keyword [en]
Animals, Antibodies; Monoclonal, Bacterial Proteins/analysis/genetics, Bacteriological Techniques, Biotinylation, Enzyme-Linked Immunosorbent Assay, Feces/microbiology, Female, Fetus/virology, Lawsonia Bacteria/*classification/genetics/immunology, Mice, Mice; Inbred BALB C, Nucleic Acid Amplification Techniques, Parvoviridae Infections/veterinary/virology, Parvovirus; Porcine/*classification/genetics/immunology, Pregnancy, Pregnancy Complications; Infectious/veterinary/virology, Sensitivity and Specificity, Swine, Swine Diseases/virology, Viral Proteins/analysis/genetics, Virion/classification/genetics, Virology/methods
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-94310DOI: 10.1373/clinchem.2005.065847PubMedID: 16723682OAI: oai:DiVA.org:uu-94310DiVA: diva2:168118
Available from: 2006-04-20 Created: 2006-04-20 Last updated: 2013-07-01Bibliographically approved
In thesis
1. Application of proximity Ligation for Detection of Proteins, Biomolecular Interactions, and Single Copies of Pathogens
Open this publication in new window or tab >>Application of proximity Ligation for Detection of Proteins, Biomolecular Interactions, and Single Copies of Pathogens
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions.

Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium.

Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 137
Molecular medicine, Proximity ligation, Cytokines, Microbial pathogens, Protein-DNA interactions, Drug screening, Molekylärmedicin
urn:nbn:se:uu:diva-6791 (URN)91-554-6531-5 (ISBN)
Public defence
2006-05-11, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15
Available from: 2006-04-20 Created: 2006-04-20Bibliographically approved

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