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| 2. |
- Gantelius, Jesper, et al.
(författare)
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Magnetic bead-based detection of autoimmune responses using protein microarrays.
- 2009
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Ingår i: New biotechnology. - : Elsevier BV. - 1871-6784. ; 26, s. 269-276
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Tidskriftsartikel (refereegranskat)abstract
- In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.
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| 3. |
- Hamsten, Carl, 1981-, et al.
(författare)
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Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC
- 2009
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Ingår i: Molecular & Cellular Proteomics. - Stanford : HighWire Press. - 1535-9476 .- 1535-9484. ; 8:11, s. 2544-2554
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Tidskriftsartikel (refereegranskat)abstract
- A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causing agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP affected cattle and controls were monitored against one third of the surface proteins of M. mycoides SC in a high-throughput magnetic bead based assay. First, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in E. coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP positive and negative sera. Signals were proven to be protein-specific by inhibition experiments and results agreed with western blot experiments. The assay's potential to monitor IgG, IgM and IgA responses over time was shown in a proof-of-concept study with 116 sera from 8 animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M mycoides SC, has been created.
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| 4. |
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| 5. |
- Larsson, Karin, et al.
(författare)
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Characterization of PrEST-based antibodies towards human Cytokeratin-17
- 2009
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 342:1-2, s. 20-32
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.
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| 6. |
- Leblanc, Neil, et al.
(författare)
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Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 155:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.
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| 7. |
- Neiman, Maja, et al.
(författare)
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Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay
- 2009
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Ingår i: Clinical and Vaccine Immunology. - Washington D.C. : American Society for Microbiology. - 1556-6811 .- 1556-679X. ; 16:11, s. 1665-1674
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Rimini, Rebecca, et al.
(författare)
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Validation of serum protein profiles by a dual antibody array approach
- 2009
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 73:2, s. 252-266
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.
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| 9. |
- Rockberg, Johan, et al.
(författare)
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Discovery of epitopes for targeting the human epidermal growth factor receptor 2 (HER2) with antibodies
- 2009
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Ingår i: Molecular Oncology. - : Wiley. - 1574-7891. ; 3:3, s. 238-247
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies have become valuable therapeutic agents for targeting of extracellular proteins in various diseases, including cancer, autoimmunity and cardiovascular disorders. For breast cancer, antibodies targeting the human HER2 have been shown to result in cell growth inhibition both in vitro and in patients with breast tumors. There is evidence to suggest that targeting multiple HER2 epitopes may result in increased growth inhibition making it interesting to find antibodies targeting new epitopes. Here, we report on a new scheme to discover antibodies directed to new epitopes using the extracellular domain of the HER2 as a model. Polyclonal antibodies were generated using recombinant protein fragments and affinity purified fractions of the antibodies were functionally characterized and precisely epitope mapped using bacterial surface display. Polyclonal antibodies towards a 127 amino acid recombinant protein fragment spanning between domains II and III of the HER2 were shown to bind to human ductal carcinoma cell line BT474 resulting in growth inhibition. Affinity purification demonstrated that antibodies to two separate regions from the N- and C-terminal end of the fragment exhibited the growth inhibition. Epitope mapping of the C-terminal antibodies revealed a 25 amino acid region (LPESFDGDPASNTAPLQPEQLQVF) with two distinct epitopes mediating efficient growth inhibition. The results suggest that antibodies directed towards this region of domain III of the HER2, distinct from the well-known monoclonal antibodies trastuzumab and pertuzumab, bind to the HER2 on living cells and exhibit growth inhibition. The work describes a new strategy to develop antibodies directed to non-overlapping epitopes and shows a path of pursuit to explore the epitope space of a target protein.
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| 10. |
- Schwenk, Jochen M., et al.
(författare)
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Antibody suspension bead arrays within serum proteomics
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:8, s. 3168-3179
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Tidskriftsartikel (refereegranskat)abstract
- Antibody microarrays offer a powerful tool to screen for target proteins in complex samples. Here, we describe an approach for systematic analysis of serum, based on antibodies and using color-coded beads for the creation of antibody arrays in suspension. This method, adapted from planar antibody arrays, offers a fast, flexible, and multiplexed procedure to screen larger numbers of serum samples, and no purification steps are required to remove excess labeling substance. The assay system detected proteins down to lower picomolar levels with dynamic ranges over 3 orders of magnitude. The feasibility of this workflow was shown in a study with more than 200 clinical serum samples tested for 20 serum proteins.
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